Methods for preventing and treating cancer using N-thiolated β-lactam compounds and analogs thereof

ABSTRACT

The subject invention concerns N-thiolated β-lactam compounds of formula A,                  
 
wherein R 1  is a hydrocarbon group having 1–8 carbon atoms; R 3  is an organothio group; and R 4  is alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, cycloalkenyl, or heterocycloalkenyl, and analogs and pharmaceutically acceptable salts, esters and amides thereof. The subject invention also concerns methods for inducing tumor cell death or inhibiting tumor cell proliferation, and methods for inducing DNA damage, inhibiting DNA replication, activating p38 MAP kinase, or activating caspase cascade activation, or releasing cytochrome C from mitochondria into the cytoplasm in a tumor cell. Methods for treating cancer using N-thiolated β-lactam compounds, as well as pharmaceutical compositions comprising the same are further disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 60/377,604, filed May 6, 2002.

FIELD OF THE INVENTION

The present invention relates to N-thiolated β-lactam compounds, andanalogs and derivatives thereof, which potently induce neoplastic cellapoptosis or inhibit neoplastic cell proliferation. Specifically, theinvention relates to methods of treating conditions characterized byabnormal cellular proliferation or dysregulation of the normal processof cell death in the cells or tissues of an animal using N-thiolatedβ-lactam compounds, and analogs and derivatives thereof.

BACKGROUND OF THE INVENTION

Apoptosis is the process by which a cell actively commits suicidethrough a tightly controlled program (See for example, Wyllie A H, etal., 1980, Cell death: the significance of apoptosis. Int Rev Cytol68:251–306). Morphologically, apoptosis is characterized by shrinkage ofthe cell, dramatic reorganization of the nucleus, active membraneblebbing, and ultimately fragmentation of the cell intomembrane-enclosed vesicles (apoptotic bodies) (Earnshaw W C, 1995,Nuclear changes in apoptosis. Curr Opin Cell Biol 7:337–43). Apoptosisoccurs in two physiological stages: commitment and execution.

Recent experiments have demonstrated that mitochondria play an essentialrole in apoptotic commitment (Green D R et al., 1998, Mitochondria andapoptosis. Science 281:1309–12). Upon apoptotic stimulation, severalimportant events occur at the mitochondria, including the release ofcytochrome C. Release of cytochrome C from the mitochondria can beinhibited by the expression of anti-apoptotic Bcl-2 family members (suchas Bcl-2 and Bcl-XL) and induced by the expression of pro-apoptoticBcl-2 proteins (such as Bax and BID). During receptor-mediatedapoptosis, BID is cleaved at its N-terminus by caspase-8. Thecarboxyl-terminal fragment of BID (MW 15 kDa) is then inserted into themembrane of the mitochondria, triggering release of mitochondrialcytochrome C (Li H, et al., 1998, Cleavage of BID by caspase-8 mediatesthe mitochondrial damage in the Fas pathway of apoptosis. Cell94:491–501).

Releasing cytochrome C from mitochondria commits the cell to die byeither apoptosis or necrosis. The cytochrome C-induced apoptotic processinvolves Apaf-1-mediated caspase activation. This cytosolic cytochrome Cinteracts with Apaf-1, which induces its association with procaspase-9,thereby triggering processing and consequent activation of caspase-9.The activated caspase-9 in turn cleaves downstream effector caspases(such as caspase-3), initiating apoptotic execution (Green et al.,supra; Martin, et al., 1995, Cell. 82:349–52; Thornberry et al., 1998,Science. 281:1312–6). It is believed that activating effector caspasesleads to apoptosis through the proteolytic cleavage of importantcellular proteins, such as poly(ADP-ribose) polymerase (PARP) (Lazebniket al., 1994, Nature. 371:346–7) and the retinoblastoma protein (RB) (Anet al., 1996, Cancer Res. 56:438–42; Janicke et al., 1996, Embo J.15:6969–78; Fattman, et al., 2001, Oncogene. 20:2918–26).

Activating the cellular apoptotic program is a current strategy fortreating human cancer. In fact, radiation and standard chemotherapeuticdrugs have been demonstrated to kill some tumor cells by inducingapoptosis (Fisher, 1994, Cell. 78:539–42).

Unfortunately, the majority of human cancers at present are resistant topresent therapies (Harrison, 1995, J Pathol. 175: 7–12; Desoize, 1994,Anticancer Res. 14: 2291–2294; Kellen, 1994, Anticancer Res.14:433–435). It is therefore essential to identify novel anti-cancercompounds that induce apoptosis. Along this line, synthetic smallcompounds have great potential to be developed into anticancer drugsbecause they can be easily synthesized and structurally manipulated forselective development.

For more than 60 years, N-thiolated β-lactam antibiotics have played anessential role in treating bacterial infections (Morin et al., Chemistryand Biology of beta-lactam Antibiotics, Vol. 1–3. New York: AcademicPress, 1982; Kukacs et al., Recent Progress in the Chemical Synthesis ofAntibiotics. Berlin, Springer-Verlag, 1990). Recently a new class ofN-thiolated β-lactam is found to inhibit bacterial growth inStaphylococcus aureus (Turos et al., 2000, Tetrahedron 56:5571–5578;Ren, et al., 1998, J. Org. Chem. 63: 8898–8917). These compounds thushave proven clinical acceptability. To date, N-thiolated β-lactamcompounds have not found use as anticancer drugs. Accordingly,N-thiolated β-lactam compounds that rapidly induce DNA damage, inhibitDNA replication, and induce an apoptotic effect, including inducing adeath program of a neoplastic cell in a time and concentration dependentmanner are desired.

BRIEF SUMMARY OF THE INVENTION

The invention concerns a method for inducing tumor cell death orinhibiting tumor cell proliferation, comprising contacting the cell withan effective amount of a N-thiolated β-lactam compound, or apharmaceutically acceptable salt, ester or amide thereof. According to apreferred embodiment, the inventive method uses N-thiolated β-lactam A-Has hereinafter defined. In a particularly preferred embodiment,N-thiolated β-lactam A is used.

The method of the present invention is preferably used to treat tumorcells present in an animal, preferably a mammal, such as a humanpatient. The tumor cells can be those of a solid tumor or a blood bornetumor. Suitable cancers that can be treated with the present inventioninclude, but are not limited to lung, breast, colon, prostate, melanoma,pancreas, stomach, liver, brain, kidney, uterus, cervix, ovaries,urinary tract, gastrointestinal, head-and-neck cancer or leukemia.N-thiolated β-lactam compounds of the present invention can beadministered to the animal orally, intramuscularly, and/ortransdermally.

The present invention also relates to pharmaceutical compositionscomprising N-thiolated β-lactam compounds of the invention. In apreferred embodiment, an N-thiolated β-lactam compound of the inventionhas the structure shown in formula (1). In one embodiment, theN-thiolated β-lactam has the structure of the compound corresponding tolactam 1 as defined in Table 1.

In yet another embodiment, the present invention relates to a method forinducing, in a tumor cell, DNA damage, DNA replication inhibition, p38MAP kinase activation, or caspase cascade activation, or cytochrome Crelease from mitochondria into the cytoplasm, comprising contacting thetumor cell with an effective amount of a N-thiolated β-lactam compound.

In a further embodiment, the present invention relates to a method forscreening an N-thiolated β-lactam compound or an analog thereof for itsability to induce tumor cell death or to inhibit tumor cellproliferation, the method comprising the steps of a) contacting aculture of tumor cells with the compound; b) preparing a lysate of thetreated cells; and c) measuring apoptosis-specific caspase-3 activity orPARP cleavage; wherein an increase in either caspase-3 activity or PARPcleavage as compared to untreated tumor cells indicates that thecompound or analog thereof is capable of inducing cancer cell death orinhibiting cancer cell proliferation. In a preferred embodiment, humanJurkat T cells can be used for screening.

The purpose and advantages of the present invention will be set forth inand apparent from the description that follows, as well as will belearned by practice of the invention. Additional advantages of theinvention will be realized and attained by the methods and compositionsparticularly pointed out in the written description and claims hereof,as well as from the appended drawings.

Both the foregoing general description and the following detaileddescription are exemplary and are intended to provide furtherexplanation of the invention claimed. Those skilled in the art willappreciate that the conception upon which this disclosure is based canreadily be utilized to prepare other N-thiolated P-lactam compounds andmethods of using such compounds for carrying out the methods of thepresent invention. It is important, therefore, that the claims beregarded as including such equivalent compounds and methods of usingsuch compounds insofar as they do not depart from the spirit and scopeof the present invention.

The accompanying drawings, which are incorporated in and constitute partof this specification, are included to illustrate and provide a furtherunderstanding of the compositions and methods of the invention. Togetherwith the description, the drawing serves to explain the principles ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows structures of eight N-thiolated β-lactam compounds of thepresent invention.

FIGS. 2A–D show the apoptosis-inducing potencies of differentN-thiolated β-lactam analogs. Jurkat T cells are treated with either 50μM of each individual compound or the vehicle DMSO (indicated by D) for8 h, followed by preparation of cellular extracts. FIGS. 2A and 2C showcell-free caspase-3 activity assay. Standard deviations are calculatedfrom three separate and independent experiments and are indicated byerror bars. FIGS. 2B and 2D show Western blot assay using a specificpolyclonal PARP antibody. The intact PARP (MW 116 kDa) and a PARPcleavage fragment (p85) are shown. Similar results are obtained in threeor more experiments.

FIGS. 3A–D show a kinetic characterization of lactam 1-inducedapoptosis. Jurkat T cells are treated with 50 μM lactam 1 for theindicated hours, followed by performance of various assays as follows.FIGS. 3A and 3D show Western blot assay using specific antibodies toPARP, BID or caspase-9. The intact caspase-9 (MW 45 kDa) and itscleavage (active) fragment (35 kDa) as well as a BID cleavage fragment(15 kDa) are shown. The experiments are conducted at least three timeswith similar results. FIG. 3B shows trypan blue incorporation assay. Thenumbers given are percentages of non-viable cells to total cells.Standard deviations are shown with error bars from a mean of at least 3different experiments. FIG. 3C shows cell-free caspase-8, caspase-9 andcaspase-3 assay. Standard deviations are shown from 3 differentexperiments.

FIG. 4 shows that lactam 1 induces cytochrome C release frommitochondria. Cytosol and mitochondria fractions are prepared fromJurkat T cells treated with 50 μM lactam 1 for indicated hours, followedby Western blot assay using a specific antibody to cytochrome C (MW 17kDa; A), the cytochrome oxidase subunit H (COX, MW 26 kDa; B) and˜-actin (MW 43 kDa; C). The unchanged levels of mitochondrial COX andcytosolic ˜3-actin protein serve as controls for equal loading andfractionation purity.

FIGS. 5A–D show that apoptosis induced by lactam 1 isconcentration-dependant. Jurkat T cells are treated with increasingconcentrations of lactam 1 for 8 hours, followed by assaying for PARPcleavage (FIG. 5A), trypan blue incorporation (FIG. 5B), and cell-freecaspase-8, caspase-9 and caspase-3 activities (FIG. 5C). Results arerepresentative of 3–5 different experiments. Standard deviations aregiven with error bars from a mean of at least 3 different experiments inB and C. FIG. 5D demonstrates that caspase inhibitors block apoptosisinduced by lactam 1. Jurkat T cells are pretreated with either aspecific inhibitor to caspase-8, caspase-9 or caspase-3 (at 25 μM), or ageneral caspase inhibitor (pan, at 25 μM), or DMSO, followed by aco-treatment with 50 μM lactam 1 for 8 hours. After that, PARP cleavageis determined in a Western blot assay.

FIGS. 6A–B show that lactam 1 dysregulates cell cycle progressionassociated with apoptosis induction. FIG. 6A shows Jurkat T cells aretreated with 50 μM lactam 1 for the indicated hours, followed by flowcytometry analysis. The cell cycle distribution is measured as thepercentage of cells that contain G₁, S, G₂ and M DNA (cells in G₁, S, G₂and M=100%). The apoptotic population is measured as the percentage oftotal cell populations with <G₁ DNA content. FIG. 6B shows Jurkat Tcells are treated with either DMSO (D) or 50 μM lactam for (1, 2, 3, or4), for five hours, followed by assaying the cell cycle distribution andSub G₁ population.

FIGS. 7A–E show that lactam 1 inhibits DNA replication and induces DNAstrand breaks in Jurkat T cells. FIGS. 7A and 7B: ³H-thymidineincorporation assay. Jurkat T cells are either untreated (as a control,indicated by C or 0 μM), or pretreated with 50 μM lactam 1 for theindicated hours (FIG. 7A), or pretreated with indicated concentrationsof lactam 1 for 2 hours (FIG. 7B). ³H-thymidine is then added, followedby a 2 hour incubation. The amount of ³H-thymidine incorporated is thenanalyzed by scintillation counting (see MATERIALS AND METHODS sectionherein). Standard deviations are shown with error bars from a mean of atleast 3 different experiments. FIGS. 7C–7E: TUNEL assay. Jurkat cellsare treated with 50 μM lactam 1 for four hours (FIG. 7D) each indicatedtime point (FIG. 7C), or with 50 μM of the indicated drug for four hours(FIG. 7E), followed by analysis of DNA strand breaks by flow cytometry(FIGS. 7C and 7E) or a fluorescence microscopy (FIG. 7D). The M1 regionrepresents the TUNEL-positive (DNA strand breaks) cells. Similar resultsare observed in 3 independent experiments.

FIGS. 8A–I show the involvement of p38 MAP kinase in lactam 1-inducedapoptosis. (FIGS. 8A–D) Jurkat T cells are treated with 50 μM lactam 1for the indicated hours (FIG. 8D), followed by Western blot assay usingspecific antibodies to phosphorylated p38 (pp38, total p38, or actin. RD(relative density) values are normalized ratios of the intensities ofpp38 or p38 band to the corresponding actin band. The experiments aredone three times with similar results. (FIGS. 8E and 8F) Jurkat T cellsare pre-treated for 1 hour with either the specific p38 MAP kinaseinhibitor PD-169316 (PD-16; at 30 μM), the pan caspase inhibitor (at 25μM), or the vehicle DMSO, followed by a co-treatment with 50 μM lactam 1for 8 h. After that, PARP cleavage is determined in Western blotting(FIG. 8E) and caspase-8, caspase-9 and caspase-3 activities are measuredin cell-free assay (FIG. 8F; and see FIG. 3). (FIG. 8G) Jurkat T cellsare pretreated with 50 μM lactam 1, 50 μM lactam 1 plus 30 μM PD16, orDMSO for 2 hours, followed by addition of ³H-thymidine. After anadditional 2 hours of incubation, the amount of ³H-thymidineincorporated is then analyzed by scintillation counting (see FIG. 7).(FIGS. 8H–I) Jurkat T cells are treated for four hours with either DMSOor lactam 1 (at 50 μM), in the absence (with DMSO) or presence of PD169316 (PD-16; at 30 μM) or Boc-D-FMK (50 μM), followed by measurementof TUNEL positivity and p38 phosphorylation as described in FIG. 7C andFIGS. 8A–8C, respectively.

FIGS. 9A–B show that lactam 1 inhibits proliferation and inducesapoptosis in four solid tumor cell lines. (FIG. 9A) MTT assay. Humanbreast (MCF-7, MDA-MB-231), prostate (PC-3), and head-and-neck (PCI-13)cancer cell lines are grown in equal cell numbers in a 24-well plate. At˜50% confluency (0 hours), three wells of each cell line are treatedwith either 50 μM lactam 1 or DMSO for 24 hours. After that, cells aresubjected to MTT assay. Standard deviations are given as described inFIG. 3. (FIG. 9B) Nuclear staining assay. MCF-7, MDA-MB231, PC-3 andPCI-13 cells are treated with 50CM lactam 1 or DMSO for 24 (MCF-7) or 48hours (other three lines), followed by collecting both detached andattached cell populations. After lactam 1 treatment, ˜50% of thesecancer cell lines became detached, whereas <5% became detached aftertreating with DMSO. Both detached and attached cell populations are usedfor nuclear staining assay with DNA staining die Hoechst 33342. Eachsample is then analyzed by fluorescence microscopy for nuclearmorphology. Similar results are obtained in 6 independent experiments.

FIG. 10 shows the proposed order of apoptotic events induced bylactam 1. A cascade of events that occur during lactam 1-inducedapoptosis is proposed based on kinetic and inhibitor studies.

FIGS. 11A–B show structures of N-thiolated β-lactam compounds of thepresent invention.

FIG. 12 shows cell-free caspase-3 activity assay. Jurkat T cells aretreated with either each indicated compound at 50 μM or the vehicle DMSOfor 8 h, followed by preparation of cellular extracts and performance ofcell-free caspase-3 activity assay.

FIGS. 13A–B show a screen for more potent analogs of the lactam 1. (FIG.13A) Structures of the N-thiolated β-lactam compounds studied. Numericaldesignations were given to each compound. (FIG. 13B) Jurkat T cells weretreated with the solvent (DMSO) or 50 μM of each indicated analog for 24h, followed by trypan blue dye exclusion assay. The numbers given arepercentages of non-viable cells to total cells. Standard deviations areshown with error bars from a mean of at least three differentexperiments.

FIGS. 14A–D show selective induction of apoptosis by lactam 1 inleukemic Jurkat T over immortalized/non-transformed NK cells. Jurkat Tand NK (YT) cells were treated with 10, 25 and 50 μM of lactam 1 for 24h (FIG. 14A) or with 30 μM of lactam 1 for indicated hours (FIGS.14B–D). (FIGS. 14A and 14B) Measurement of PARP cleavage in Western blotassay. The intact PARP (116 kDa) and a PARP cleavage fragment (p85) areshown. (FIG. 14C) Trypan blue dye exclusion assay. The numbers given arepercentages of non-viable cells to total cells. Standard deviations areshown with error bars from a mean of at least three differentexperiments. (FIG. 14D) Morphological changes of Jurkat T and YT cellsafter treatment. Photographs under a phase-contrast microscope (100×).

FIGS. 15A–D show dose-response comparison between Jurkat T and YT cellstreated with lactam 12 and lactam 1 to induce cell apoptosis. Jurkat T(FIGS. 15A and 15B) and YT cells (FIGS. 15C and 15D) were treated with2, 10, 25, and 50 μM of lactam 12 versus 50 μM of lactam 1 for either 12(FIGS. 15B and 15D) or 24 h (FIGS. 15A and 15C), followed by trypan blueexclusion (FIGS. 15A and 15C) or Western blot assay using anti-PARPantibody (FIGS. 15B and 15D). Results are representative of threedifferent experiments. Standard deviations are shown with error barsfrom a mean of at least three independent experiments (FIGS. 15A and15C).

FIGS. 16A–B show a kinetic comparison between lactam 12 and lactam 1 toinduce apoptosis in Jurkat T cells. Jurkat T cells were treated with 25μM of lactam 12 versus 50 μM of lactam 1 for 3, 6, 12, and 24 h,followed by trypan blue dye exclusion assay (FIG. 16A), and PARPcleavage in Western blot assay (FIG. 16B). Results are representative ofthree different experiments. Standard deviations are shown with errorbars from a mean of at least three independent experiments.

FIGS. 17A–B show lactam 12 induces sub-G₁ cell population andTUNEL-positivity. Jurkat T cells (0 h) were treated with 50 μM of lactam12 for the indicated hours. (FIG. 17A) Measurement of sub-G₁ DNA contentby flow cytometry analysis. The percentage of sub-G₁ cell populationrepresents the cell populations with DNA fragmentation. (FIG. 17B)Measurement of DNA strand breaks by TUNEL assay. The numbers indicatethe percentage of TUNEL-positive population. Results of representativeof three independent experiments are shown.

FIGS. 18A–C show effects of β-lactams on caspase activation and colonyformation. (FIG. 18A) Prostate cancer LNCaP cells were treated for 48 hwith 2, 10, and 25 μM of lactam 12 versus 50 μM of lactam 1. Cell-freecaspase-3 activity was then determined by incubating whole cell extractswith caspase-3 substrate and measuring free AMCs. (FIGS. 18B and 18C)LNCaP cells were plated in soft agar with the solvent DMSO or 50 μM ofthe indicated β-lactams. Cells were then cultured for 21 days withoutaddition of new drug. The plates were scanned and a representative wellfrom each treatment was selected for presentation (FIG. 18B). Colonieswere quantified with an automated counter and presented as mean valuesfrom triplicate independent experiments. Error bars denote standarddeviations (FIG. 18C).

FIGS. 19A–D show lactam 1 selectively inhibits proliferation and induceapoptosis in SV40-transformed human fibroblasts, but not the normal,parental human fibroblasts. The normal (WI-38) and SV40-transformed(VA-13) human fibroblasts, grown in either 96-well plates (FIG. 19A, 0h) or 60-mm dishes (FIGS. 19B–D, 0 h), are treated with either 50 μMlactam 1 or DMSO (or D) for up to 48 hours. (FIG. 19A) MTT assay. (FIG.19B) Cell-free caspase-9 activity assay. A protein extract (20 μg) isincubated with 20 μM Ac-LEHD-AFC (the specific caspase-9 substrate) in a96-well plate at 37° C. for 2 h. After incubation, the liberatedflorescent AFC groups are measured by Wallac Victor 1420 Multilabelcounter with 405/535 nM filters. FIG. 19C shows processing andactivation of caspase-3 detected in Western blot assay. Both intact andactive forms of caspase-3 are indicated. FIG. 19D shows nuclear stainingassay. Treatment with lactam 1 or DMSO for 48 hours induced ˜60% and<5%, respectively, of VA-13 cells detached. When WI-38 cells are treatedwith either lactam 1 or DMSO for 38 hours, no detachment is observed.Both detached and attached populations of Va-13 or WI-38 cells arecollected, and used for nuclear staining assay with DNA staining dyeHoechst 33342.

DETAILED DISCLOSURE OF THE INVENTION

Reference will now be made in detail to the present preferredembodiments of the invention, examples of which are illustrated in theaccompanying figures. The methods and corresponding compositions of theinvention will be described in conjunction below.

An important property of candidate anticancer drugs is the ability toinduce tumor cell apoptosis (Pfundt et al., 2001, J Pathol. 193:248–55).The present invention concerns N-thiolated β-lactam compounds thatinduce apoptosis and inhibit cell cycle progression in several humancancer cell lines.

Definitions

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'SPHARMACEUTICAL SCIENCES, 15th Ed., Mack Publ. Co., Easton, Pa. (1975).

A “hyperproliferative disorder” is any condition in which a localizedpopulation of proliferating cells in an animal is not governed by theusual limitation of normal growth. Examples of hyperproliferativedisorders include tumors, neoplasms, lymphomas and the like.

“Pharmaceutically acceptable salt, ester or amide” as used herein,relates to a chemical modification of a compound of the presentinvention wherein the chemical modification takes place either at afunctional group of the compound or on the aromatic ring.Pharmaceutically acceptable salt, ester or amide, include anypharmaceutically acceptable salt, amide, ester, or other derivative. Thederivative of a compound of the present invention, upon administrationto a recipient, is capable of providing, directly or indirectly, acompound of this invention or an active metabolite or residue thereof.Particularly favored derivatives increase the bioavailability of thecompounds of this invention when such compounds are administered to amammal (e.g., by allowing an orally administered compound to be morereadily absorbed into the blood) or enhance delivery of the parentcompound to a biological compartment (e.g., the brain or lymphaticsystem).

As known to those of skill in the art, “salts” of the compounds of thepresent invention can be derived from inorganic or organic acids andbases. Examples of acids, for purposes of illustration and notlimitation, include hydrochloric, hydrobromic, sulfuric, nitric,perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic,succinic, p-toluenesulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic andbenzenesulfonic acids. Other acids, such as oxalic, while not inthemselves pharmaceutically acceptable, can be used to prepare saltsthat are useful intermediates in obtaining the compounds of theinvention and their pharmaceutically acceptable acid addition salts.

Examples of bases, for purposes of illustration and not limitation,include alkali metal (e.g. sodium) hydroxides, alkaline earth metal(e.g., magnesium) hydroxides, and ammonia. Examples of salts, forpurposes of illustration and not limitation, include: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate and undecanoate. Fortherapeutic use, salts of the compounds of the present invention will bepharmaceutically acceptable. However, salts of acids and bases that arenon-pharmaceutically acceptable can also find use, for example, in thepreparation or purification of a pharmaceutically acceptable compound.

β-Lactam Compounds

The subject invention concerns N-thiolated β-lactam compounds and usesthereof. Compounds of the invention have apoptotic and/oranti-proliferative properties against tumor cells.

β-lactam compounds encompassed within the scope of the present inventioncan have the general formula A:

wherein R₁ is a hydrocarbon group having 1–8 carbon atoms and includesalkyl, alkenyl, and alkynyl groups;

R₃ is an organothio group; and

R₄ is an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, cycloalkenyl, or heterocycloalkenyl, any of which canbe optionally substituted with R₂, wherein R₂ is one or more halides,hydroxyl, nitro, cyano, alkyl, alkenyl, alkynyl, aryl, heteroaryl,cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, alkoxy,amido, amino, carboxylic ester group, —CHO, —COOH, or COX, wherein X isCl, F, Br, or I;

or a pharmaceutically acceptable salt, ester or amide thereof.

In one embodiment, β-lactam compounds of the present invention can havethe following formula I:

in which R₁ is a hydrocarbon group having 1–8 carbon atoms and includesalkyl, alkenyl, and alkynyl groups; R₂ is one or more halides, hydroxyl,nitro, cyano, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, alkoxy, —CHO, —COOH,amido, amino, carboxylic ester group, or COX, wherein X is Cl, F, Br, orI; and R₃ is an organothio group; or a pharmaceutically acceptable salt,ester or amide thereof.

In exemplified embodiments, the β-lactam compounds of formula I havestructures shown in FIGS. 1, 11, and 13A, and Table 1 below.

TABLE 1 N-thiolated β-lactam Compounds of formula I of the Invention R₂(position on the phenyl ring) Compound # R₁ 2 3 4 5 6 R₃ lactam 1 —CH₃—Cl —S—CH₃ lactam 2 —CH₃ —Cl —H lactam 3 —CH₃ —Cl —S—CH₂CH₃ lactam 4—CH₃ —Cl —S—(CH₂)₃ CH₃ lactam 5 —CH₃ —Cl —S—CH₃ lactam 6 —CH₃ —Cl —S—CH₃lactam 7 —CH₃ —Cl —S—CH₂—Ph lactam 8 —CH₃ —CO₂CH₃ —S—CH₃ lactam 9 —CH₃—Cl —S—CH₂—CH₃ lactam 10 —CH₃ —Cl —S—(CH₂)₃CH₃ lactam 11 —CH₃ —Cl—S—(CH₂)₇CH₃ lactam 12 —CH₃ —Cl SCH₂CO₂CH₃ lactam 13 —COCH₃ —Br —S—CH₃lactam 14 —COCH₃ —F —S—CH₃ lactam 15 —CH₃ —Br —S—CH₃ lactam 16 —CH₃ —Br—S—CH₃ lactam 17 —CH₃ —CO₂Me —S—CH₃ lactam 18 —CH₃ —CO₂CH₂CH₃ —S—CH₃lactam 19 —CH₃ —CO₂(CH₂)₄CH₃ —S—CH₃ lactam 20 —CH₃ —Cl H lactam 21 —CH₃OH H lactam 22 —CH₃ OH —S—CH₃ lactarn 23 —CH₃ —Br —S—CH₃ lactam 24 —CH₃—F —S—CH₃ lactam 25 —CH₃ —F —S—CH₃ lactam 26 —CH₃ —F —S—CH₃ lactam 27—CH₃ —F —F —S—CH₃ lactam 28 —CH₃ —F —F —S—CH₃ lactam 29 —CH₃ —F —F—S—CH₃ lactam 30 —CH₃ —F —F —S—CH₃ lactam 31 —CH₃ —F —F —S—CH₃ lactam 32—CH₃ —F —Cl —S—CH₃ lactam 36 —Ph —NO₂ —S—CH₃ lactam 42 —CH₃ —Cl—S—CH₂—Ph lactam 44 —CH₃ —CH₃ —Ph OCH₃ lactam 45 —CH₃ —CN —Ph OCH₃lactam 46 —CH₃ —I —S—CH₃ lactam 47 —CH₃ —NO₂ —S—CH₃ lactam 48 —CH₃—S—CH₃ lactam 49 —CH₃ —F —S—CH₃ Ph = phenyl

β-lactam compounds encompassed within the scope of the present inventioncan have the general formula B:

wherein R₁ is a hydrocarbon group having 1–8 carbon atoms and includesalkyl, alkenyl, and alkynyl groups;

R₃ is an organothio group;

R₄ is an aryl, heteroaryl, cycloalkyl, heterocycloalkyl, cycloalkenyl,or heterocycloalkenyl, any of which can be optionally substituted withR₂ wherein R₂ is one or more halides, hydroxyl, nitro, cyano, alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl,cycloalkenyl, heterocycloalkenyl, alkoxy, amido, amino, carboxylic estergroup, —CHO, —COOH, or COX, wherein X is Cl, F, Br, or I; and

(N) is, preferably a straight or branched 2–4 carbon alkyl, alkenyl, oralkynyl chain connecting the β-lactam ring to the R₄ group;

or a pharmaceutically acceptable salt, ester or amide thereof.

β-lactam compounds of the present invention can also have the followingformula II:

in which R₁ is a hydrocarbon group having 1–8 carbon atoms; R₂ is one ormore halide, hydroxyl, nitro, cyano, alkyl, alkenyl, alkynyl, aryl,heteroaryl, cycloalkyl, heterocycloalkyl, cycloalkenyl,heterocycloalkenyl, alkoxy, amido, amino, carboxylic ester group, —CHO,—COOH, or COX, wherein X is Cl, F, Br, or I; (N) is, preferably astraight or branched 2–4 carbon alkyl, alkenyl, alkynyl, or alkynylchain connecting the β-lactam ring to the phenyl; and R₃ is anorganothio group; or a pharmaceutically acceptable salt, ester or amidethereof.

TABLE 2 N-thiolated β-lactam Compounds of formula II of the Invention R₂(position on the phenyl ring) Compound # R₁ 2 3 4 R₃ lactam 33^(a) —CH₃—S—CH₃ lactam 34^(b) —CH₃ —S—CH₃ lactam 37^(c) —CH₃ —S—CH₃ lactam 38^(c)—CH₃ —S—(CH₂)₂OH lactam 39^(c) —CH₃ —S—cyclohexyl lactam 40^(c) —CH₃—S—Ph lactam 41^(c) —CH₃ —S—CH₂—Ph ^(a)where N = —(CH₂)₂— ^(b)where N =—(CH)₂— ^(c)where N = —C≡C— Ph = phenyl

β-lactam compounds of the present invention can also have the structureof Formula III:

in which R₁ is a hydrocarbon group having 1–8 carbon atoms; R₂ is one ormore halide, hydroxyl, nitro, cyano, alkyl, alkenyl, alkynyl, aryl,heteroaryl, cycloalkyl, heterocycloalkyl, cycloalkenyl,heterocycloalkenyl, alkoxy, amido, amino, carboxylic ester group, —CHO,—COOH, or COX, wherein X is Cl, F, Br, or I; and R₃ is an organothiogroup; or a pharmaceutically acceptable salt, ester or amide thereof.

It will be appreciated by those skilled in the art that compounds of theinvention having one or more chiral center(s) can exist in and beisolated in optically active and/or racemic forms. Some compounds canexhibit polymorphism. It is well known in the art how to prepareoptically active forms (for example, by resolution of the racemic formby recrystallization techniques, by synthesis from optically-activestarting materials, by chiral synthesis, or by chromatographicseparation using a chiral stationary phase). It is to be understood thatthe present invention encompasses any racemic, optically-active,polymorphic, or stereoisomeric form, or mixtures thereof, of thecompounds of the invention and methods of using such compounds asdescribed herein.

As used herein, the term “alkyl” refers to a straight or branched chainalkyl moiety. Included within this group are, for example, methyl,ethyl, n-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isoamyl, hexyl, and octyl.

The term “alkenyl” refers to a straight or branched chain alkyl moietyhaving in addition one double bond. Included within this group are, forexample, ethenyl, propenyl, 1- and 2-butenyl, pentenyl, and hexenyl.

The term “alkynyl” refers to a straight or branched chain alkyl moietyhaving in addition one triple bond. Included within this group are, forexample, ethynyl, propynyl, 1- and 2-butynyl, pentynyl, and hexynyl.

The term “alkoxy” refers to an alknyl ether moiety wherein the termalkyl is as defined above. Included within this group are, for example,methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy,sec-butoxy, and tert-butoxy.

The term “acyl” refers to an alkyl or aryl group bonded through acarbonyl (R—C(O)—) group. Included within this group are, for example,acetyl and benzoyl.

The term “cycloalkyl” refers to a saturated alicyclic moiety, andincludes benzofused cycloalkyls. Included within this group are, forexample, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, and decalinyl. The term “benzofused cycloalkyl” refers to abenzene ring sharing a common bond with the ring of a cycloalkyl.Included within this group are, for example, indanyl andtetrahydronaphthyl.

The term “cycloalkenyl” refers to an alicyclic moiety having in additionone double bond. Included within this group are, for example,cyclopentenyl and cyclohexenyl.

The term “heterocycloalkyl” refers to a heterocyclic moiety having oneor more heteroatoms selected from the group of N, O, and S, and includesbenzofused heterocycloalkyls. Included within this group are, forexample, pyrrolidinyl, pyrrolyl, piperidinyl, and morpholinyl. The term“benzofused heterocycloalkyl” refers to a benzene ring sharing a commonbond with the ring of a heterocycloalkyl. Included within this groupare, for example, indolinyl and tetrahydroquinolinyl.

The term “heterocycloalkenyl” refers to an alicyclic moiety having oneor more heteroatoms selected from the group of N, O, and S, and havingin addition one double bond. Included within this group is, for example,dihydropyranyl.

The term “aryl” refers to a homocyclic aromatic moiety that can be asingle ring or multiple rings which are fused together or linkedcovalently. Included within this group are, for example, phenyl,indenyl, biphenyl, naphthyl, anthracenyl, and phenathracenyl group.

The term “heteroaryl” refers to an aromatic ring system of five to tenatoms of which at least one atom is selected from the group N, O and S.Included within this group are, for example, pyrolyl, furyl, thienyl,imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridyl, pyrrimidinyl,indolyl, quinolinyl, and isoquinolinyl.

Preparation of the N-thiolated β-Lactam Compounds

N-thiolated β-lactam compounds of the present invention can besynthesized according to the procedure given in Ren et al., 1998, J.Org. Chem. 63:8898–8917, which is incorporated herein by reference inits entirety. It will be apparent to one of ordinary skill in the art inthe field of the present invention how the starting compounds andprocedures below can be easily modified to produce other desiredcompounds, both those described herein, and others such as R₂ being oneor more halogens in meta, para or ortho position on phenyl ring.

As can be understood with reference to Reaction Scheme 1 (below), whilemost antibiotics require demanding multi-step syntheses orsemi-synthetic procedures to reach the final active substance,N-thiolated β-lactams such as 5 can be prepared in a single step fromn-protio lactams 4 using a published procedure (Shah, N. V.; Cama, L. D.Synthesis of a Novel Carbapenem-Potassium(5R,6R)-1,1-Difluoro-2-phenyl-6-(1R-hydroxyethyl)-carbapeN-2-em-3-carboxylate.The Use of a New N-Protecting Group in β-lactam Synthesis. Heterocycles1987, 25, 221). Although some of N-thiolated β-lactams are commerciallyavailable, most variants of the N-thiolated β-lactams can be prepared inonly two chemical steps. The first step is a Staudinger coupling of anacid chloride 1 with an N-(4-methoxyphenyl) imine 2, followed byN-dearylation of β-lactam 3 with ceric ammonium nitrate. After that, thealkylthio group SR³ is attached to the lactam nitrogen using an easilyprepared phthalimide reagent. Yields for each of these steps aretypically above 90% on a multi-gram scale regardless of the nature ofthe R¹, R² and R³ groups. The compounds are generally obtained inpurified form by crystallization directly from the crude reaction media,omitting the need for column chromatography or HPLC. The structure andpurity of all compounds are determined by ¹H and ¹³C NMR spectroscopy,infrared spectroscopy, mass spectrometry, and elemental analysis. Usingthis simple three-step procedure (see Reaction Scheme 1, below),N-thiolated β-lactams can be synthesized.

The following general experimental procedures can be employed. All air-or moisture-sensitive procedures are performed under an argon atmosphereusing glassware and syringes that are pre-dried in an oven overnight at120° C., and assembled while still hot. The imines are prepared byheating equimolar amounts of the appropriate aldehyde and amine inrefluxing benzene solution in the presence of a small amount ofp-toluenesulfonic acid under Dean-Stark conditions, followed byfiltering the cooled solution through an approximately one inch plug ofsilica gel to remove residual amine. The purity of the crude imine ischecked by ¹H NMR prior to use. The acid chlorides are synthesizedaccording to standard protocols by heating the corresponding carboxylicacid in thionyl chloride, removing residual volatiles by distillation,and used without further purification. THF and Et₂O are distilledimmediately prior to use from sodium/benzophenone under argon, andCH₂Cl₂ is freshly distilled from CaH₂ under N₂. Reactions are followedby TLC with fluorescence indicator (SiO₂-60, F-254) or 1% aqueous KMnO₄stain. Flash chromatography is performed using 40 μm silica gel. ¹H NMRspectra are recorded at 300, 360, 400 or 500 MHz and ¹³C NMR spectra areobtained at 75, 100, or 125 MHz. IR spectra are obtained as a thin filmsmeared onto NaCl plates. Mass spectra are run using electron impact orchemical ionization methods.

Methods of Treatment

As noted above, the present invention provides methods of treatingconditions that arise as the result of dysregulation of the normalprocess of cell death in the cells or tissue of a subject. Dysregulationof the cell death process is associated with many conditions. Suchconditions include tumors, cancers, neoplasms, autoimmune disorders(e.g., systemic lupus erythematosus, rheumatoid arthritis,graft-versus-host disease, Sjogren's syndrome and myasthenia gravis);hyperproliferative disorders (such as B or T cell lymphoma,neuroblastoma, and chronic lymphocytic leukemia), chronic inflammatoryconditions (such as psoriasis, asthma, or Crohn's disease), otherconditions such as osteoarthritis and atherosclerosis, and those inducedby DNA and/or RNA viral infections, wherein the viruses include, but arenot limited to, herpes virus, papilloma virus and human immunodeficiencyvirus (HIV).

In neoplasms, for example, normal cell death is inhibited, allowinghyperproliferative growth of cells. Aberrant functioning of normal celldeath can also result in serious pathologies including autoimmunedisorders, viral infections, conditions induced by viral infections,neurodegenerative diseases, and the like. The present invention providesmethods of treating these and other conditions. Thus, the subjectinvention also concerns methods for treating or preventinghyperproliferative disorders in a patient. These disorders are treatedby administering an effective amount of a β-lactam compound of thepresent invention. These β-lactam compounds are therapeuticallyeffective on their own.

Conditions that benefit from treatment with the β-lactam compounds ofthe present invention share the common etiology of dysregulation of thecell death process. Normal apoptosis occurs via several pathways, witheach pathway having multiple steps. The methods described herein areuseful in treating dysregulated apoptosis and necrosis. Without beinglimited by one theory, the effective β-lactam compounds described hereininduce or promote cell death when the cell death process ismalfunctioning. Thus, in addition to treating conditions associated withdysregulated apoptosis, the compounds of this invention also treatconditions in which there is not any apoptotic defect. For example, incertain viral infections, while there is not any apoptotic defect, celldeath can be promoted by inducing necrosis.

One aspect of the subject invention concerns methods for inducing tumorcell death or inhibiting tumor cell proliferation by contacting orexposing a tumor cell to an N-thiolated β-lactam compound of the presentinvention. In a further aspect, the present invention concerns methodsfor inducing DNA damage, inhibition of DNA replication, p38 MAP kinaseactivation, caspase cascade activation, and/or mitochondrial cytochromeC release into cytoplasm in a tumor cell comprising contacting orexposing the tumor cell to an N-thiolated β-lactam compound of thepresent invention. In one embodiment of the present methods, the tumorcell is from a mammal. In a preferred embodiment, the tumor cell is froma human.

The subject invention also concerns methods for treating or preventingcancer in a patient, wherein the method comprises administering to thepatient an effective amount of an N-thiolated β-lactam compound of thepresent invention. The subject method can be used to treat or preventcancers including, but not limited to, lung cancer, breast cancer, coloncancer, prostate cancer, melanomas, pancreatic cancer, stomach cancer,liver cancer, brain cancer, kidney cancer, uterine cancer, cervicalcancer, ovarian cancer, cancer of the urinary tract, gastrointestinalcancer, head-and-neck cancer, or leukemia. In one embodiment of thesubject method, the N-thiolated β-lactam compound is administeredorally, intramuscularly, and/or transdermally. Preferably, the patientbeing treated is a mammal, and more preferably, the mammal is a human.

To “treat” as intended herein, means to induce cell death (wherein thecell death is either apoptotic or necrotic) in cells or tissue which arecausative (primary or distal) of the disorder being treated. Forexample, in hyperproliferative disorders, the methods will treat thedisorder by inducing apoptosis of the hyperproliferative cells, such asneoplastic cells. In this embodiment, reduction in tumor size or tumorburden is one means to identify that the object of the method has beenmet. In other aspects, treatment encompasses restoration of immunefunction or regulation of immune dysfunction, as in autoimmune disordersand chronic inflammatory conditions.

Methods of Identifying Potential Therapeutic Agents

Also provided herein is an assay to screen for β-lactam compounds aspotential agents to effectively treat conditions associated with thedysregulation of the apoptotic or necrotic pathway.

The method comprises contacting a dysregulated cell, i.e., a cellaffected by the disorder, such as a tumor cell or otherhyperproliferative condition, with an effective amount of a β-lactamcompound to be screened. In a further aspect of this invention, anuntreated control cell is further assayed and compared to the tumor celltreated with a β-lactam compound.

A preferred method for screening a β-lactam compound or an analogthereof for its ability to induce tumor cell death or to inhibit tumorcell proliferation comprises: contacting a culture of tumor cells withthe β-lactam compound, preparing a lysate of the β-lactam treated cells,and measuring apoptosis-specific caspase-3 activity or PARP cleavage;and relating the measured apoptosis-specific caspase-3 activity or PARPcleavage to the β-lactam ability to induce tumor cell death or toinhibit tumor cell proliferation wherein an increase in either caspase-3activity or PARP cleavage as compared to untreated tumor cells indicatesthat the compound or analog thereof is capable of inducing cancer celldeath or inhibiting cancer cell proliferation. A skilled artisan willreadily recognize that any tumor or neoplastic cell line is suitable forscreening and a variety of assays can be used. For example, the tumorcell can be a leukemia cell or a solid tumor cell.

To identify these potential β-lactam therapeutic agents, appropriateassay conditions (e.g., incubation time, temperature, culturemaintenance medium, etc.) can be readily determined by one of skill inthe art, some of which are exemplified in the Examples below.

In a further aspect, the N-thiolated β-lactam compounds of thisinvention are further characterized and identified by their ability toinduce caspase-8, caspase-9 and caspase-3 activation, S-phase arrest,DNA strand breaks, or p38 MAP kinase activation.

Use of β-Lactam Compounds for Preparing Medicaments

The β-lactam compounds of the present invention are also useful in thepreparation of medicaments to treat a variety of conditions associatedwith dysregulation of cell death as described above. Thus, one of skillin the art would readily appreciate that any one or more of thecompounds described more fully below, including the many specificembodiments, can be used by applying standard pharmaceuticalmanufacturing procedures to prepare medicaments to treat the manydisorders described herein above. Such medicaments can be delivered tothe subject by using delivery methods that are well-known in thepharmaceutical arts.

Compositions and Formulations

Although β-lactam compounds of the present invention can be administeredalone, β-lactam compounds can also be administered as a pharmaceuticalformulation comprising at least one additional active ingredient,together with one or more pharmaceutically acceptable carriers therefor.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to thepatient.

Formulations include those suitable for oral, rectal, nasal, topical(including transdermal, buccal and sublingual), vaginal, parenteral(including subcutaneous, intramuscular, intravenous and intradermal) andpulmonary administration. The formulations can conveniently be presentedin unit dosage form and can be prepared by any methods well known in theart of pharmacy. Such methods include the step of bringing intoassociation the active ingredient with the carrier which constitutes oneor more accessory ingredients. In general, the formulations are preparedby uniformly and intimately bringing into association the activeingredient with liquid carriers or finely divided solid carriers orboth, and then if necessary shaping the product.

Formulations of the present invention suitable for oral administrationcan be presented as discrete units such as capsules, cachets or tablets,each containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient can also bepresented as bolus, electuary, or paste.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions for topical administration according to thepresent invention can be formulated as an ointment, cream, suspension,lotion, powder, solution, paste, gel, spray, aerosol or oil.Alternatively, a formulation can comprise a patch or a dressing such asa bandage or adhesive plaster impregnated with active ingredients, andoptionally one or more excipients or diluents. Formulations suitable fortopical administration to the eye also include eye drops wherein theactive ingredient is dissolved or suspended in a suitable carrier,especially an aqueous solvent for the agent.

Formulations for rectal administration can be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate. Formulations suitable for vaginal administration can bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing in addition to the agent, such carriers as areknown in the art to be appropriate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of about 20 to about 500 microns which is administered in themanner in which snuff is taken, i.e., by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid for administrationas, for example, nasal spray, nasal drops, or by aerosol administrationby nebulizer, include aqueous or oily solutions of the agent.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which can containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which can include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. The formulations can be presented in unit-dose ormulti-dose sealed containers, such as for example, ampoules and vials,and can be stored in a freeze-dried (lyophilized) condition requiringonly the addition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions can be prepared from sterile powders, granules andtablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate fractionthereof, of an agent. It should be understood that in addition to theingredients particularly mentioned above, the formulations of thisinvention can include other agents conventional in the art regarding thetype of formulation in question. For example, formulations suitable fororal administration can include such further agents as sweeteners,thickeners, and flavoring agents. It also is intended that the agents,compositions, and methods of this invention be combined with othersuitable compositions and therapies.

Pharmaceutical Delivery

Various delivery systems are known and can be used to administer atherapeutic agent of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, receptor-mediated endocytosis (see, e.g.Wu et al., 1987, J. Biol. Chem. 262:4429–4432), and the like. Methods ofdelivery include, but are not limited to, intra-arterial, intramuscular,intravenous, intranasal, and oral routes. In a specific embodiment, thepharmaceutical compositions of the invention can be administered locallyto the area in need of treatment; such local administration can beachieved by, for purpose of illustration and not limitation, localinfusion during surgery, by injection, or by means of a catheter.

Therapeutic amounts can be empirically determined and will vary with thepathology being treated, the subject being treated, and the efficacy andtoxicity of the agent. Similarly, suitable dosage formulations andmethods of administering the agents can be readily determined by thoseof skill in the art.

The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and can take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They can also take the form ofsuspensions, solutions, and emulsions of the active ingredient inaqueous or nonaqueous diluents, syrups, granulates or powders. Inaddition to an agent of the present invention, the pharmaceuticalcompositions can also contain other pharmaceutically active compounds ora plurality of compounds of the invention.

Ideally, the agent should be administered to achieve peak concentrationsof the active compound at sites of the disease. Peak concentrations atdisease sites can be achieved, for example, by intravenously injectingof the agent, optionally in saline, or orally administering, example, atablet, capsule or syrup containing the active ingredient.

Kits

Furthermore, the invention also comprehends a kit wherein N-thiolatedβ-lactam compounds of the present invention are provided. The kit caninclude a separate container containing a suitable carrier, diluent orexcipient. The kit can also include additional anti-cancer, anti-tumoror antineoplastic agent, antioxidant, DNA topoisomerase II enzymeinhibitor or an inhibitor of oxidative DNA damage or antimicrobial, orcyclo-oxygenase and/or lipoxygenase, NO or NO-synthase non-steroidalanti-inflammatory, apoptosis and platelet aggregation modulating orblood or in vivo glucose modulating agent and/or an agent which reducesor alleviates ill effects of antineoplastic, anti-tumor or anti-canceragents, antioxidant, DNA topoisomerase II enzyme inhibitor orantimicrobial, or cyclo-oxygenase and/or lipoxygenase, NO orNO-synthase, apoptosis, platelet aggregation and blood or in vivoglucose modulating and/or non-steroidal anti-inflammatory agents for co-or sequential-administration. The additional agent(s) can be provided inseparate container(s) or in admixture with the inventive polyphenolcompounds. Additionally, the kit can include instructions for mixing orcombining ingredients and/or administration.

From the above description of the invention, one of skill in the artreadily understands that the various methods of treatment, diagnosticmethods, use of compounds to prepare medicaments, delivery of suchmedicaments, and the making of the compounds, can be practiced in manydifferent ways, as exemplified by the many examples presented below.

Abbreviations used herein include Apaf-1, apoptotic protease-activatingfactor 1; PARP, poly(ADP-ribose) polymerase; MAP, mitogen-activatedprotein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; Ab, antibody; COX, cytochrome oxidase unit II; TUNEL, terminaldeoxynucleotidyl transferase-mediated UTP nick-end labeling; Z-IETD-AFC,N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-7-amino-4-trifluoromethyl coumarin;Ac-LEHDAFC, N-acetyl-Leu-Glu-His-Asp-7-amino-4-trifluoromethyl coumarin;Ac-DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin;Ac-IETD-CHO, N-acetyl-Ile-Glu-Thr-Asp-CHO (aldehyde);Z-LE(OMe)HD(OMe)-FMK,N-benzyloxycarbonyl-Leu-Glu(OMe)-His-Asp(OMe)-fluoromethyl ketone;Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde); Boc-D-FMK,N-tert-butoxycarbonyl-Asp-fluoromethyl ketone; PD 169316,4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole; PBS,phosphate-buffered saline; TdT, terminal deoxynucleotidyl transferase;SAR, structure-activity relationship; DMSO, dimethyl sulfoxide; MT-21, apreviously reported synthetic γ lactam.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Synthesis of N-thiolated β-lactam Compounds

N-thiolated β-lactam compounds can be synthesized as shown in thereaction scheme below. See: Staudinger, 1907, Liebigs Ann. Chem. 356:51,Georg et al., 1993, in: “The Organic Chemistry of β-lactams”, VerlagChemie: New York, pp. 295–368.

Procedure for the Preparation of N-aryl Protected β-Lactams

To a stirred solution of Et₃N (1.25 mL, 9.0 mmol) and imine (7, R═CCPh)(1.88 g, 8.0 mmol) in CH₂Cl₂ (75 mL) at room temperature is added viacannula a solution of methoxyacetyl chloride (6, X═OMe) (0.91 g, 10.0mmol) in CH₂Cl₂ (20 mL). The reaction mixture is stirred at roomtemperature for 30 min, poured into 5% aqueous HCl (75 mL), andextracted with CH₂Cl₂ (3×50 mL). The combined organic layers are driedover MgSO₄, filtered, and evaporated to give a brown oil that slowlycrystallizes upon standing. Flash chromatography (2:1 CH₂Cl₂:hexanes andthen CH₂Cl₂) of the crude material affords 2.2 g (89%) of β-lactam (8,X═OMe, R═CCPh): white solid; 116–117 C; ¹H NMR (400 MHz, CDCl₃) δ7.54–7.35 (m, 7H), 6.9 (d, J=7.8 Hz, 2H), 4.96 (d, J=4.8 Hz, 1H), 4.81(d, J=4.8 Hz, 1H), 3.78 (s, 3H), 3.70 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ163.1, 157.4, 132.6, 129.3, 129.2, 129.1, 128.8, 119.1, 115.0, 84.8,81.7, 59.1, 56.1, 56.0, 50.3; IR (thin film) 1752 cm⁻¹ (β-lactam C═O).Anal. Calcd for C₁₉H₁₇NO₃: C, 74.25; H, 5.58; N, 4.56. Found: C, 74.10;H, 5.60; N, 4.57.

Procedure for the Dearylation of N-aryl β-Lactams.

To a solution of N-p-methoxyphenyl β-lactam (8, X═OMe, R═CCPh) (2.2 g,7.2 mmol) in CH₃CN (100 mL) at 0° C. is added 100 mL of an aqueoussolution of ammonium cerium(IV) nitrate (11.8 g, 21.6 mmol) over 5 min.The reaction mixture is stirred for 25 min and then poured into aqueous5% NaHSO₃ (100 mL), and the aqueous mixture is extracted with Et₂O (3×50mL). The combined organic layers are treated with 5% NaHCO₃ (100 mL),and the aqueous layer is back-washed with one portion of diethyl ether(50 mL). The combined organic layers are dried over MgSO₄, filtered, andevaporated. Flash chromatography of the crude mixture affords 1.28 g(89%) of (9, X═OMe, R═CCPh, R′═H): white solid (mp 121–122° C.); 1H NMR(400 MHz, CDCl₃) δ 7.42–7.35 (m, 5H), 6.80 (broad s, 1H), 4.72 (d, J=4.8Hz, 1H), 4.60 (d, J=4.8 Hz, 1H), 3.60 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ167.5, 132.4, 129.4, 128.9, 122.7, 87.9, 87.1, 83.7, 58.8, 46.6; HRMS(CI, isobutane) calcd for C₁₂H₁₁NO₂ (M+1) 202.0865, obsd 202.0884. Anal.Calcd for C₁₂H₁₁NO₂: C, 71.63; H, 5.51; N, 6.96. Found: C, 71.55; H,5.53; N, 6.91.

Procedure for the N-methylthiolation of β-Lactams.

To a solution of (9, X═OMe, R═CCPh, R′═H) (1.28 g, 6.4 mmol) in THF at−78° C. is added n-butyllithium (5.0 mL, 1.38 M in hexanes, 6.9 mmol).After 30 minutes, methyl methanethiolsulfonate (0.85 g, 6.6 mmol) isadded and the reaction mixture is stirred for 12 hours with warming toroom temperature. The mixture is poured into 5% aqueous NH₄Cl (50 mL)and extracted with CH₂Cl₂ (3×50 mL). The combined organic layers aredried over MgSO₄, filtered, and evaporated. Flash chromatography of thecrude mixture affords 1.26 g (80%) of N-methylthio compound (9, X═OMe,R═CCPh, R′═SMe): colorless solid; mp 74–76 C; ¹H NMR (400 MHz, CDCl₃) δ7.42 (d, J=8.8 Hz, 2H), 7.30 (m, 3H), 4.72 (d, J−4.8 Hz, 1H), 4.63 (d,J=4.8 Hz, 1H), 3.56 (s, 3H), 2.58 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ169.8, 132.4, 129.6, 129.0, 122.5, 89.3, 86.6, 82.5, 59.0, 55.1, 22.7;IR (thin film) 1772 cm⁻¹ (β-lactam C═O). HRMS (CI, isobutane) calculatedfor C₁₃H₁₃NO₂S (M+1) 248.0742, obsd 248.0734. Anal. calculated forC₁₃H₁₃NO₂S: C, 63.13; H, 5.30. Found: C, 63.08; H, 5.33.

MATERIALS AND METHODS FOR EXAMPLES 2–7

Materials.

Fetal calf serum, propidium iodide, MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], trypanblue, and RNase A are purchased from Sigma (St. Louis, Mo.). RPMI 1640,Dulbecco's modified Eagle's medium, penicillin and streptomycin arepurchased from Life Technologies, Inc. (Rockville, Md.). Polyclonalantibodies to human PARP is from Boehringer Mannheim (Indianapolis,Ind.); to caspase-8 (Ab-1) from Oncogene Research Products (Boston,Mass.). Monoclonal antibodies to Tyr-182 phosphorylated and total p38protein are from Santa Cruz Biotechnology (Santa Cruz, Calif.); tocaspase-9 (Ab-2) and caspase-3 (Ab-1) are from Oncogene ResearchProducts; to cytochrome C from BD PharMingen (San Diego, Calif.); tocytochrome oxidase unit 11 (COX) from Molecular Probes (Eugene, Oreg.).Goat antibody to actin and anti-rabbit IgG-horseradish peroxidase arefrom Santa Cruz Biotechnology. The APO-DIRECT Kit for TUNEL staining ispurchased from BD PharMingen. [methyl-³H]Thymidine is obtained fromAmersham Pharmacia (Piscataway, N.J.). Z-IETD-AFC (the specificcaspase-8 substrate), Ac-LEHD-AFC (the specific caspase-9 substrate),Ac-DEVD-AMC (the specific caspase-3 substrate), Ac-IETD-CHO (thespecific caspase-8 inhibitor), Z-LE(OMe)HD(OMe)-FMK (the specificcaspase-9 inhibitor), Ac-DEVD-CHO (the specific caspase-3 inhibitor),Boc-D-FMK (a pan caspase inhibitor), and PD169316 (the specific p38 MAPkinase inhibitor) are obtained from Calbiochem (San Diego, Calif.).

Synthesis of β-Lactams.

β-Lactams 1–7 (FIG. 1) are prepared as racemates (with cisstereochemistry) using a procedure described previously (Ren et al.,1998; Turos et al., 2000).

Cell Cultures, Protein Extraction and Western Blot Assay.

Human Jurkat T cells were cultured in RPMI 1640, supplemented with 10%fetal calf serum, 100 units/ml of penicillin and 100 μg/ml ofstreptomycin. Human breast cancer MCF-7 and MDA-MB-231 cells, humanprostate cancer PC-3 cells, and human head-and-neck cancer PCI-13 cellswere grown in Dulbecco's modified Eagle's medium containing 10% fetalcalf serum, penicillin and streptomycin. All the cell lines weremaintained in a 5% CO₂ atmosphere at 37° C. A whole cell extract wasprepared as described previously (An et al., 1998). Briefly, cells areharvested, washed with PBS and homogenized in a lysis buffer (50 mMTris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, 0.5 mM PMSF, and0.5 mM dithiothreitol) for 30 min at 4° C. After that, the lysates arecentrifuged at 14,000×g for 30 min and the supernatants are collected aswhole cell extracts. Equal amounts of protein extract (50 μg) areresolved by SDS-polyacrylamide gel electrophoresis and then transferredto a nitrocellulose membrane (Schleicher & Schuell, Keene, N.H.) using aSemi-Dry Transfer System (BIO-RAD; Hercules, Calif.). The enhancedchemiluminescence (ECL) Western Blot analysis is then performed usingspecific antibodies to the proteins of interest.

Cell-Free Caspase Activity Assay.

Cell-free caspase activities were determined by measuring the cleavageof AMC or AFC groups from each respective caspase substrate, asdescribed for example by Nam S. et al., 2001, Ester bond-containing teapolyphenols potently inhibit proteasome activity in vitro and in vivo. JBiol Chem 276:13322–30 with some modifications. Briefly, a preparedprotein extract (20 μg) is incubated in a buffer containing 50 mMTris/pH 8.0 along with each respective caspase substrate at 20 μM in a96 well plate. The reaction mixture is incubated at 37° C. for 2 h.After incubation, the liberated fluorescent AMC or AFC groups aremeasured by a Wallac Victor 1420 Multilabel counter (Turku, Finland)with 355/460 nM and 405/535 nM filters, respectively.

Trypan Blue Assay.

The trypan blue exclusion assay was performed by injecting 10 μl of cellsuspension containing 0.2% trypan blue dye into a hemoicytometer andcounting. Numbers of cells that absorbed the dye and those that excludedthe dye are counted, from which the percentage of non-viable cell numberto total cell number is calculated.

Subcellular Fractionation.

Both cytosolic and mitochondria fractions were isolated at 4° C. using aprotocol by Gao G and Dou Q P, 2000, N-terminal cleavage of bax bycalpain generates a potent proapoptotic 18-kDa fragment that promotesbcl-2-independent cytochrome C release and apoptotic cell death. J CellBiochem 80:53–72 with some modifications. At each time point, cells werewashed twice with PBS, resuspended in a hypotonic buffer containing 20mM HEPES (pH 7.5), 1.5 mM MgCl₂, 5 mM KCl and 1 mM DTT, and incubated onice for 10 min. The cells are dounced 30 times, and the lysate iscentrifuged at 2,000×g for 10 min. The supernatant is collected andcentrifuged again at the same condition. The resulting supernatant isthen centrifuged at 20,500×g for 30 min, followed by collection of boththe supernatant (cytosol) and pellet fractions. The pellet is washedtwice with a buffer containing 210 mM mannitol, 70 mM sucrose, 5 mMTris-HCl (pH 7.5) and 1 mM EDTA, and resuspended in the lysis buffer asthe mitochondria fraction.

Flow Cytometry.

Cell cycle analysis based on DNA content was performed as described byAn B, et al. (1998) Novel dipeptidyl proteasome inhibitors overcomeBcl-2 protective function and selectively accumulate thecyclin-dependent kinase inhibitor p27 and induce apoptosis intransformed, but not normal, human fibroblasts. Cell Death Differ5:1062–75. At each time point, cells are harvested, counted, and washedtwice with PBS. Cells (5×10⁶) are suspended in 0.5 ml PBS, fixed in 5 mlof 70% ethanol for at least 2 h at −20° C., centrifuged, resuspendedagain in 1 ml of propidium iodide staining solution (50 μg propidiumiodide, 100 units RNase A and 1 mg glucose per ml PBS), and incubated atroom temperature for 30 minutes. The cells are then analyzed withFACScan (Becton Dickinson Immunocytometry, Calif.), ModFit LT and WinMDIV.2.8 cell cycle analysis software (Verity Software; Topsham, Me.). Thecell cycle distribution is shown as the percentage of cells containingG₁, S, G₂, and M DNA judged by propidium iodide staining. The apoptoticpopulation is determined as the percentage of cells with sub-G₁ (<G₁)DNA content.

³H-Thymidine Incorporation Assay.

Incorporation of ³H-thymidine into cells was measured as disclosed inSmith D M and Dou Q P, 2001, Green tea polyphenol epigallocatechininhibits DNA replication and consequently induces leukemia cellapoptosis. Int J Mol Med 7:645–52. Jurkat T cells are pre-treated with aselected lactam for the indicated number of hours, followed byco-incubation with 2 μl/ml of [methyl-³H]-thymidine [80 Ci (1.5TBq)/mMol] at 37° C. for 2 hours. After harvesting, the cell pellet iswashed with PBS, resuspended in 0.5 ml of PBS and collected on a glassmicrofiber filter. The filter is then washed with 5 ml/filter of PBS,followed by 5 ml/filter of ice-cold 0.1N NaOH and 5 ml/filter ofethanol. The filters containing fixed DNA are dried, and the remainingradioactivity is measured on a scintillation counter.

TUNEL Assay.

Terminal deoxynucleotidyl transferase-mediated UTP nick end labeling(TUNEL) was performed to determine the extent of DNA strand breaks.TUNEL assay was performed with an APO-Direct kit per the manufacturer'sinstructions. In brief, cells are fixed in 1% paraformaldehyde andethanol at 20° C. overnight and then permeabilized with Proteinase K.After permeabilization, Fluorescein conjugated dNTP's and TdT enzyme(Terminal Deoxynucleotidyl Transferase) are added to the cells. The TdTenzyme is then able to label free ends of DNA with Fluoresceinconjugated dNTPs that could then be detected by flow cytometry. For thefluorescence microscopy of TUNEL-positive cells, Jurkat T cells werelabeled and analyzed in accordance with the manufacturer's instructions(see for example, An et al., 1998, supra).

MTT Assay.

MCF-7, MDA-MB-231, PC-3 and PCI-13 cells were grown to 50% confluency ina 24 well plate. Triplicate wells of cells were then treated with 50 μMlactam 1 for 24 hours. A stock 5 mg/ml of MTT in serum-free media wasthen added to the cell cultures at a final concentration of 1 mg/ml,followed by a 3 hour incubation at 37° C. After cells crystallized, themedia was removed and DMSO added to dissolve the metabolized MTTproduct. The absorbance was then measured on a Wallac Victor 1420Multilabel counter at 540 nM.

Nuclear Staining Assay.

To assay nuclear morphology, both the detached or remaining attachedsolid tumor cells were washed with PBS, fixed with 70% ethanol for 1hour, and stained with Hoechst 33342 (50 μM) for 30 minutes. The nuclearmorphology of cells was visualized by a fluorescence microscope.

EXAMPLE 2 Screen for Apoptotically Active β-Lactams

A library of β-lactam analogs was screened for their ability to induceapoptosis. A representative group of 7 compounds and their structures isshown in FIG. 1. The screening procedure is accomplished by treatinghuman Jurkat T cells with each compound at 50 μM for 8 hours. This isfollowed by preparation of cell lysates and measurement ofapoptosis-specific caspase-3 activation (by cell-free caspase-3 activityassay) and PARP cleavage (by Western blotting).

Among the tested compounds, lactam 1 was found to have the greatestpotency to induce caspase-3 activation and PARP cleavage within 8 hoursof treatment (FIGS. 2A and 2B). Several important structure-activityrelationships (SARs) were observed. First, the N-methylthio group isrequired for the apoptosis-inducing activity of lactam 1. Lactam 2,which is an analog of lactam 1 that lacks the N-methylthio group (FIG.1), induces neither caspase-3 activation nor PARP cleavage (FIGS. 2A and2B, lactam 2 vs. lactam 1). In fact, lactam 2-treated cells showed nomorphological changes, similar to that observed for DMSO(vehicle)-treated cells (FIGS. 2A and 2B, lactam 2 vs. D, data notshown).

The second SAR observed was that an increase in the number of carbons onthe N-thio group was inversely proportional to the apoptosis-inducingability of these β-lactams. An increase from one carbon (lactam 1) totwo carbons (lactam 3) in this chain decreased ˜50% of caspase-3activity and PARP cleavage (FIGS. 2A and 2B, lactam 1 vs. lactam 3). Afurther increase to four carbons on the N-thio group (lactam 4) causes˜65% decrease in the apoptosis-inducing activity (FIGS. 2A and 2B,lactam 4 vs. lactam 1). Replacement of the N-methylthio with aN-benzylthio group (FIG. 1, lactam 7) also decreased theapoptosis-inducing activity by ˜70% (FIGS. 2A and 2B, lactam 1 vs.lactam 7).

Another SAR was associated with the chlorophenyl group in lactam 1.Lactams 1, 5, and 6 are isomers with the chlorine group at ortho-, meta-and para-positions, respectively, on the phenyl ring (FIG. 1). Althoughboth lactams 5 and 6 have similar potency in inducing caspase-3 activityand PARP cleavage, both of them are less potent than lactam 1 (by ˜20%;FIGS. 2A and 2B). Based on these results, lactam 1 was chosen as a leadcompound for further apoptosis and cell cycle studies.

EXAMPLE 3 Lactam 1-Induced Apoptosis is Caspase-Dependent and Associatedwith Cytochrome C Release

Lactam 1-induced apoptosis was studied by performing both kinetics andconcentration-response experiments. When Jurkat T cells were treatedwith 50 μM lactam 1 for 2, 4, 6, 8, 12 or 24 hours, apoptosis occured ina time-dependent manner (FIGS. 3A and 3B). The PARP cleavage fragmentp85 appears after 4 hours of treatment and its levels increaseafterwards (FIG. 3A). Associated with this, the non-viable cellpopulation, as determined by a trypan blue exclusion assay, increased by20% at 4 hours, and further increased to 60% after 24 hours of treatmentwith lactam 1 (FIG. 3B).

Activation of caspase-8, caspase-9 and caspase-3 was measured by bothcell-free activity assay (FIG. 3C) and Western blot analysis (FIG. 3D)to determine which caspases are activated during lactam 1-inducedapoptosis. The caspase-8 activity was detected at 2 hours and later timepoints, with a maximal level at 6 hours (FIG. 3C). Western blot assayconfirms cleavage and activation of caspase-8 at 2 hours with peakingamounts of caspase-8 fragment at 6 hours (MW 18 kDa; FIG. 3D).Consistent with caspase-8 activation, a 15-kDa fragment of Bid (Li etal., 1998) was observed as early as 2 hours after lactam 1 treatment,and peaks at 6 hours (FIG. 3D). The activity of caspase-9 is firstdetected at 4 hours and then increases afterwards (FIG. 3C). Theincreased level of the caspase-9 activity was associated with increasedlevels of the active caspase-9 fragment (MW 35 kDa; FIG. 3D). However,caspase-9 activity levels (by enzyme activity assay) and cleavagefragment amounts (by Western blot) were lower than those detected forcaspase-8 (FIGS. 3C and 3D). The cell-free caspase-3 activity was alsoobserved first at 4 hours and dramatically increased after 6 hourstreatment (FIG. 3C), in very similar kinetics to that of caspase-3cleavage detected by Western blotting (FIG. 3D). Furthermore,kinetically, caspase-3 activation is parallel to PARP cleavage (FIGS. 3,3C, 3D vs. A), which agrees with the observation that caspase-3 isresponsible for cleaving PARP (Lazebnik Y A, Kaufmann S H, Desnoyers S,Poirier G G and Earnshaw W C, 1994, Cleavage of poly(ADP-ribose)polymerase by a proteinase with properties like ICE. Nature 371:346–7).Lactam 1-induced apoptosis is associated with activation of thesecaspases.

It has been shown that MT-21, a synthetic compound with a β-lactam ring(a different class of structures from the β-lactams studied here) isable to induce mitochondrial cytochrome C release and apoptotic celldeath (Watabe M, Machida K and Osada H, 2000, MT-21 is a syntheticapoptosis inducer that directly induces cytochrome c release frommitochondria. Cancer Res 60:5214–22). Whether lactam 1 is able to inducecytochrome C release from the mitochondria was examined. In anexperiment similar to that associated with FIG. 3, Jurkat T cells weretreated with lactam 1 for up to 12 hours, followed by isolation ofcytosolic and mitochondrial fractions and measurement of the cytochromeC levels (FIG. 4). High levels of mitochondrial cytochrome C weredetected in untreated cells, associated with low levels of cytosoliccytochrome C (FIG. 4A). After 2 to 4 hours treatment with lactam 1,levels of mitochondrial cytochrome C decreased while those of cytosoliccytochrome C significantly increased (FIG. 4A), indicating release ofcytochrome C from the mitochondria. Although the mitochondrialcytochrome C levels further decreased after 6 hours or longer treatment,little or no cytochrome C is detected in the cytosol (FIG. 4A),suggesting loss of cytosolic cytochrome C in the later stages ofapoptosis (compare to FIG. 3B). The observed cytochrome C release frommitochondria to the cytosol is not an artifact as constitutive levels ofthe mitochondria-specific COX (FIG. 4B) and the cytosolic β-actinprotein (FIG. 4C) were also observed. Release of cytochrome C beganprior to activation of caspase-3 (FIGS. 4 vs. 3).

Jurkat T cells were then treated for 8 hours with various concentrationsof lactam 1 (FIG. 5). Induction of apoptosis-specific PARP cleavage wasdependent on the concentrations of lactam 1 used. Low levels of p85 PARPfragment were detected when 20 μM lactam 1 is used, which furtherincreased using 30 and 40 μM, and significantly increased using 50 μM.At 60 μM lactam 1 caused almost complete degradation of both the intactPARP protein and the p85 PARP fragment (FIG. 5A). Loss of membranepermeability was also found, a late event in apoptosis (Earnshaw W C.1995, Nuclear changes in apoptosis. Curr Opin Cell Biol 7:337–43; WyllieA H, Kerr J F and Currie A R, 1980, Cell death: the significance ofapoptosis. Int Rev Cytol 68:251–306), and was also lactam1-concentration-dependent: ˜10% at 20–40 μM, ˜30% at 50 μM, and ˜80% at60 μM (FIG. 5B).

When a cell-free caspase activity assay was performed, activation ofcaspase-8, caspase-9 and caspase-3 was also found to depend onconcentrations of lactam 1 (FIG. 5C). Compared to lysates of untreatedcells (0 μM), the levels of caspase-8 increased by 2-fold when 30 to 40μM lactam-1 was used, and by 5-fold when 50 μM lactam 1 is used (FIG.5C). Levels of caspase-3 are increased by 2-, 3-, 5- and 11-fold whenlactam 1 is used at 20, 30, 40 or 50 μM, respectively (FIG. 5C). Higherlevels of caspase-9 activity were also detected in lysates of cellstreated with higher concentrations of lactam 1, although caspase-9activity levels were lower than those detected for caspase-8 andcaspase-3 (FIG. 5C).

Both caspase activation and apoptosis induction were observed in time-and concentration-dependent fashions (FIGS. 3 and 5), indicating thatcaspases are required for lactam 1-induced apoptotic cell death. JurkatT cells were pre-treated for 1 hour with an individual caspaseinhibitor, a general caspase inhibitor (pan), or the vehicle (DMSO),followed by a co-treatment for 8 hours with 50 μM lactam 1. Pre- andco-incubation with each of the caspase inhibitors completely blockedlactam 1-induced PARP cleavage (FIG. 5D) and apoptosis-associatedmorphological changes (data not shown). Therefore, activation of thecaspases is required for lactam 1-induced apoptosis.

EXAMPLE 4 Lactam 1-Induced Apoptosis is Associated with an Increased SPhase Population

It has been suggested that dysregulation of cell cycle progression isinvolved in the initiation of apoptosis (Lee S, Christakos S and Small MB, 1993, Apoptosis and signal transduction: clues to a molecularmechanism. Curr Opin Cell Biol 5:286–91; Dou Q P, 1997, Putative rolesof retinoblastoma protein in apoptosis. Apoptosis 2:5–8; Smith D M, etal., 2000, Regulation of tumor cell apoptotic sensitivity during thecell cycle (Review). Int J Mol Med 6:503–7). The cell cycle distributionof Jurkat T cells that had been treated with lactam 1 is measured in thesame kinetics (FIG. 3) and concentration-response (FIG. 5) experimentsto determine whether lactam 1-induced apoptosis is associated with cellcycle-specific changes.

In the kinetics experiment, a slight decrease in G₁ and a correspondingincrease in S phase population (2–3%) were observed after lactam 1treatment for 2 to 4 hours (FIG. 6A). This was accompanied by inductionof apoptotic cell death, as measured by increased apoptotic sub-G₁ (<G₁)cell population (2%; FIG. 6A) and PARP cleavage (FIG. 3A). After 6 to 12hours treatment, S phase population was increased by up to 13%, whilethat of G₁ further decreased, without any apparent change in G₂/Mpopulation (FIG. 6A). Increased levels of sub-G₁ population (5–16%; FIG.6A) and PARP cleavage (FIG. 3A) were also observed. A 24 hour treatmentwith lactam 1 further increased S (20%) and sub-G₁ (30%) populations(FIG. 6A). Concentration-response experiments (data not shown) providedfurther support that lactam 1-induced apoptosis is associated withincreased S phase population.

In screening apoptotically active β-lactams, it was found that compounds2, 4, 3 and 1 increased cellular apoptosis in a stepwise fashion (FIG.2). Whether these lactams also cause S phase accumulation in a similarmanner was examined. Treatment of Jurkat T cells with 50 μM of lactam 2for 5 hours did not induce accumulation of either S or sub-G₁populations, similar to that of DMSO-treated cells (FIG. 6B, lactam 2vs. D). In contrast, under the same conditions, treatment with lactams4, 3 and 1 increased S phase population by 8%, 15% and 21%, respectively(FIG. 6B), associated with stepwise increases in sub-G₁ apoptoticpopulations of 2%, 10% and 14%, respectively (FIG. 6B). This dataindicates that the number of carbons bound to the N-thio group is notonly important for its apoptosis-inducing activity but also for itsability to arrest cells in S phase.

EXAMPLE 5 Lactam 1 Inhibits DNA Replication, Associated with Inductionof DNA Damage

To determine whether the increased S phase population by lactam 1 is dueto inhibition of DNA replication, a ³H-thymidine incorporation assay wasperformed with or without lactam 1 in both kinetics andconcentration-response experiments. In the kinetics experiment, Jurkat Tcells were pre-treated with 50 μM lactam 1 or DMSO for 0, 2, 4, 6 or 8h, followed by a 2 hour co-treatment with ³H-thymidine. After that,cells were harvested and the amount of incorporated radioactive³H-thymidine determined. When both lactam 1 and ³H-thymidine are addedat the same time and then co-incubated for 2 h, incorporation of the³H-thymidine was inhibited by ˜70%, compared to the control cells (FIG.7A, 0 h vs. C). A pre-incubation with lactam 1 for 2 to 8 h caused 95%inhibition of ³H-thymidine incorporation. Thus, lactam 1 inhibited³H-thymidine incorporation, and did so immediately after itsadministration. The fact that lactam 1 inhibited ³H-thymidineincorporation within such a short time period (2 hours) indicates thatlactam 1 is directly affecting the ability of the cell to replicate itsDNA and this effect is not due to a change in cell cycle (see FIG. 6A).

In the concentration-response experiment, Jurkat T cells werepre-incubated for 2 h with various concentrations of lactam 1, followedby a 2 hour co-incubation with ³H-thymidine (a total treatment length of4 h). Inhibition of ³H-thymidine incorporation was found to depend onlactam 1 concentrations used: 20% inhibition at 20 μM, 45% at 30 μM, 90%at 40 μM, and ˜100% at 50 or 60 μM. The half-maximal inhibition valuefor incorporation of ³H-thymidine (IC₅₀) in intact Jurkat cells wasdetermined to be 32 μM.

To determine whether lactam 1 could induce DNA damage that would lead tothe inhibition of DNA replication observed (FIGS. 7A and B), which wouldthen be responsible for blockage of S phase progression (FIG. 6) andinduction of apoptosis (FIGS. 1–6; also see FIG. 10), a TUNEL assay wasimplemented that detects DNA strand breaks, and the TUNEL-positive cellsare either quantified by flow cytometry or observed under fluorescencemicroscopy. Treatment with lactam 1 for 1 hour did not induce DNA strandbreaks, as compared to the untreated cells (0 h) that stained negativefor nick-end labeling (FIG. 7C, 0 h vs. 1 h). However, after just 2 hourincubation with lactam 1, more than half of the cell population hadshifted into the M1 region which demonstrated a positive signal for DNAstrand breaks (FIG. 7C, 2 h). At this time, the S phase population isonly slightly increased (compare FIG. 6A) and apoptosis had not beeninitiated (FIG. 3A). After a 4 hour treatment with lactam 1, almost theentire population of Jurkat cells contain damaged DNA, as shown by bothflow cytometry (FIG. 7C) and fluorescence microscopy (FIG. 7D). Underthe same conditions, the S population slightly increases (FIG. 6A) andapoptosis just starts to be detectable (FIG. 3A). These results suggestthat lactam 1 induces DNA strand breaks prior to S phase accumulationand apoptosis induction. The fact that lactam 1 induces DNA damage inthe entire cell population within 4 hours (FIGS. 7C and 7D) alsoindicates that it acts via a cell cycle-independent manner.

Lactams 2, 4, 3, and 1 were tested to see if the ability to induceapoptosis (FIGS. 13 and 18) and S phase accumulation (FIG. 6) matchedthat of their DNA-damaging abilities. After 4 hour treatment, DMSO orlactam 2 did not cause any DNA damage, while lactams 4 and 3 induced DNAdamage in 3 and 16% of the cell population, respectively (FIG. 7E).Again, treatment with lactam 1 for 4 h causes nearly 100% of the treatedcell population to become TUNEL-positive (FIG. 7E). As a comparison, thetraditional DNA damaging agent etoposide (also referred to herein asVP-16) at the same concentration induced only 10% of the cells to becomeTUNEL-positive (FIG. 7E).

EXAMPLE 6 p38 MAP Kinase Activation is Necessary for Lactam 1-InducedApoptosis

It has been shown that multiple stimuli including DNA damaging agentsinduce apoptosis via activation of p38 MAP kinase (Kummer J L, et al.,1997, Apoptosis induced by withdrawal of trophic factors is mediated byp38 mitogen-activated protein kinase. J Biol Chem 272:20490–4;Sanchez-Prieto R, et al., 2000, A role for the p38 mitogen-acitvatedprotein kinase pathway in the transcriptional activation of p53 ongenotoxic stress by chemotherapeutic agents. Cancer Res 60:2464–72).Whether lactam 1 could activate p38 MAP kinase during apoptosisinduction was examined, because lactam 1 is able to induce DNA strandbreaks (FIGS. 7C and 7D). In this experiment, Jurkat T cells weretreated with 50 μM lactam 1 for up to 12 hours, followed by measuringlevels of phosphorylated (the activated form of p38 MAP kinase) andtotal p38 protein in Western blot assay. The levels of Tyr-182phosphorylated p38 protein were increased by 3-fold at 2 h and reachedits maximum (˜9-fold) by 6 h (FIG. 8A). In has been shown that dualphosphorylation of p38 on Tyr-182 and Thr-180 activates this kinase(Raingeaud J, et al., 1995, Pro-inflammatory cytokines and environmentalstress cause p38 mitogen-activated protein kinase activation by dualphosphorylation on tyrosine and threonine. J Biol Chem 270:7420–6). Incontrast, the levels of total p38 protein remained relatively unchanged(FIG. 8B). Therefore, lactam 1-induced DNA damage triggered activationof p38 before S population accumulation and apoptosis induction.

Lactam 4 showed a 2.6 fold normalized increase in phosphorylation of p38over that of the control (FIG. 8D). Lactam 3 showed a further activationof p38 with a 3.2 fold induction over the control. Again, lactam 1induced maximal p38 phosphorylation with a 5.9 fold increase (FIG. 8D).

To determine whether p38 activation is necessary for the apoptoticeffects elicited by lactam 1, Jurkat T cells were pre-treated witheither PD-169316, a specific p38 kinase inhibitor (Kummer et al., 1997),or the vehicle DMSO for 1 hour, followed by a co-treatment with lactam 1for 8 hours. Pre- and co-treatment with PD-169316 completely inhibitedthe process of PARP cleavage induced by lactam 1, as compared to thecontrol cells (FIG. 8E). In addition, PD-169316 potently inhibitedlactam 1-induced activation of caspase-8, caspase-9 and caspase-3, asmeasured by cell-free caspase activity assay (FIG. 8F). In fact, thecaspase-inhibitory effects of the p38 kinase inhibitor are comparable tothose of the pan caspase inhibitor (FIG. 8F).

Whether PD169316 could potentially inhibit caspase activity directly wasalso investigated. Jurkat T cells were treated with 50 μM VP-16 for 5hours, followed by preparation of cell lysates and measurement ofcell-free caspase activities, in the absence or presence of PD169316 orthe pan caspase inhibitor. The results show that PD169316 did notinhibit caspase-8, caspase-9 or caspase-3 activities in theVP-16-treated cell preparation. In contrast, the pan caspase inhibitorcompletely blocked the VP-16-induced caspase activities (data notshown). This result suggests that during lactam 1-induced apoptosis, p38activation occurs upstream of caspase activation and is necessary forcaspase-mediated cell death.

Given that lactam 1 is able to inhibit DNA replication (FIG. 7) andinduce apoptosis (FIGS. 1–6) and that lactam 1-induced apoptosis can beblocked by PD-169316 (FIGS. 8E and 8F), the ability of the p38 inhibitorto affect the DNA replication-inhibitory activity of lactam 1 wasstudied. An ³H-thymidine incorporation assay was performed using Jurkatcells treated with lactam 1 alone or a combination of lactam 1 andPD-169316. Inhibition of DNA replication by lactam 1 was not affected byaddition of PD-169316 (FIG. 8G). Thus, the p38 kinase inhibitorinhibited the lactam 1-induced downstream apoptotic events, but did notaffect the ability of lactam 1 to inhibit DNA replication.

To further investigate the order of lactam 1-induced apoptotic events,effects of PD-169316 and the pan caspase inhibitor Boc-D-FMK on TUNELpositivity and p38 phosphorylation were measured. In this experiment,growing Jurkat T cells (control) were treated for 4 hours with lactam 1in the absence (with DMSO) or presence of PD169316 or Boc-D-FMK,followed by measurement of TUNEL-positive cells and phosphorylated p38levels. Again, Lactam 1 treatment induced 96% TUNNEL positivity.Similarly, cells that had been co-treated with lactam 1 and PD169316 orBoc-D-FMK, show 95% and 97% TUNNEL positivity, respectively (FIG. 8H),demonstrating that neither the p38 inhibitor nor the pan caspaseinhibitor could block DNA strand breaks induced by lactam 1. This dataalso suggests that DNA damage must lie upstream of p38 and caspaseactivation. In addition, lactam 1-induced p38 phosphorylation was notaffected by Boc-D-FMK (FIG. 8I), supporting the conclusion that p38activation occurs upstream of caspase activation (compare to FIG. 8F).

EXAMPLE 7 Lactam 1 Inhibits Cell Proliferation and Induces Apoptosis inSeveral Solid Tumor Cell Lines

The effects of lactam 1 on several other human solid tumor cell linesare studied. Exponentially grown (0 hour) human breast (MCF-7,MDA-MB-231), prostate (PC-3), and head-and-neck (PCI-13) cancer celllines were treated with either 50 μM lactam 1 or DMSO for 24 hours,followed by performance of an MTT assay which measures the status ofcell viability and thus cell proliferation. The DMSO-treated cellscontinued to proliferate after 24 hours (FIG. 9A). However, aftertreatment with lactam 1, cellular viability of MCF-7 cells, MDA-MB-231cells and PCI-13 cells decreased by 80% and that of PC-3 cells decreasedby 60% (FIG. 9A).

To determine whether lactam 1-mediated growth inhibition is due to celldeath, these tumor cell lines were treated with 50 μM lactam 1 or anequal percentage of DMSO, followed by separation of the attached anddetached cell populations. Both attached and detached cell populationswere then used for detection of apoptotic nuclear changes. After a 24hour treatment with lactam 1, ˜50% of MCF-7 cells became detached. Allthe detached MCF-7 cells exhibited typical apoptotic nuclearcondensation and fragmentation (FIG. 9B). The cellular detachment ismost likely triggered by apoptosis induction, because the remainingattached MCF-7 cells also show apoptotic nuclear morphology (FIG. 9B).Little or no detachment was observed in MCF-7 cells treated with DMSO;consistent with that, all the remaining attached cells contain normal,round nuclei (FIG. 9B). Similar to MCF-7 cells, about half ofMDA-MB-231, PC-3 and PCI-13 cells become detached after a 48 hourtreatment with lactam 1 but not DMSO. Almost all the detached cellsexhibited an apoptosis-specific nuclear morphology (FIG. 9B). These datademonstrate that lactam 1 is able to inhibit cell proliferation andinduce cell death in these breast, prostate, and head and neck solidtumor cell lines.

MATERIALS AND METHODS FOR EXAMPLES 8–12

Materials.

Fetal bovine serum (Tissue Culture Biologicals, Tulane, Calif,),3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),Dimethyl sulfoxide (DMSO) and trypan blue were purchased fromSigma-Aldrich (St. Louis, Mo.). RPMI 1640, Dulbecco's modified Eagle'smedium (DMEM), MEM non-essential amino acids solution, MEM sodiumpyruvate solution, penicillin, and streptomycin were purchased fromInvitrogen (Carlsbad, Calif.). Polyclonal antibody to human PARP wasobtained from Roche Molecular Biochemicals (Indianapolis, Ind.). TheAPO-DIRECT kit for terminal deoxynucleotidyl transferase-mediated UTPnick-end labeling (TUNEL) staining was purchased from BD Pharmingen (SanDiego, Calif.).

Synthesis of β-Lactams.

The β-lactam analogs (FIG. 13) were prepared as racemates (with cisstereochemistry) using a procedure described previously (Ren X F, etal., 1998, Studies on nonconventionally fused bicyclic beta-lactams. JOrganic Chem 63:8898–17; Turos E, Konaklieva M I, Ren R X F, Shi H,Gonzalez J, Dickey S, Lim D V., 2000, N-thiolated bicyclic andmonocyclic beta-lactams. Tetrahedron 56:5571–78).

Cell Culture, Protein Extraction, and Western Blot Assay.

Human Jurkat T cells and human prostate cancer LNCaP cells were culturedin RPMI 1640 medium, supplemented with 10% fetal bovine serum, 100units/ml penicillin, and 100 μg/ml streptomycin. Human YT cells werecultured in RPMI 1640 medium supplemented with 1 mM MEM sodium pyruvatesolution, 0.1 mM MEM non-essential amino acids solution, 10% fetalbovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Allcell lines were maintained at 37° C. in a humidified incubator with anatmosphere of 5% CO₂. A whole-cell extract was prepared as describedpreviously (An B, Goldfarb R H, Siman R, Dou Q P., 1998, Noveldipeptidyl proteasome inhibitors overcome Bcl-2 protective function andselectively accumulate the cyclin-dependent kinase inhibitor p27 andinduce apoptosis in transformed, but not normal, human fibroblasts. CellDeath Differ 5:1062–75). Briefly, cells were harvested, washed with PBSand homogenized in a lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA,150 mM NaCl, 0.5% NP-40, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5mM dithiothreitol) for 30 min at 4° C. Afterwards, the lysates werecentrifuged at 12000×g for 15 min at 4° C. and the supernatantscollected as whole-cell extracts. Equal amounts of protein extract (60μg) were resolved by SDS-polyacrylamide gel electrophoresis andtransferred to a nitrocellulose membrane (Schleicher & Schuell, Keene,N.H.) using a Semi-Dry Transfer System (Bio-Rad, Hercules, Calif.). Theenhanced chemiluminescence Western blot analysis was then performedusing specific antibodies to the proteins of interest.

Trypan Blue Assay.

The trypan blue dye exclusion assay was performed by mixing 20 μl ofcell suspension with 20 μl of 0.4% trypan blue dye before injecting intoa hemocytometer and counting. The number of cells that absorbed the dyeand those that exclude the dye were counted, from which the percentageof nonviable cell number to total cell number was calculated.

Morphological Assessment of Apoptosis.

To assess morphological changes of cells, 50 μl of treated or untreatedcell suspension were transferred to a glass slide at the indicated timepoints. The slides were observed under a phase-contrast microscope(Leica; Wetzlar, Germany) and photographs were taken (100×). Apoptoticcells were identified by their distinct morphological changes.

TUNEL Assay.

Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)was used to determine the extent of DNA strand breaks (Smith D M, Dou QP., 2001, Green tea polyphenol epigallocatechin inhibits DNA replicationand consequently induces leukemia cell apoptosis. Int J Mol Med7:645–52). The assay was performed following manufacturer's instructionusing the APO-Direct kit. In brief, the harvested cells were fixed in 1%paraformaldehyde for 15 min on ice, washed with PBS, and then fixedagain in 70% ethanol at −20° C. overnight. The cells were then incubatedin DNA labeling solution (containing terminal deoxynucleotidyltransferase (TdT) enzyme, fluorescein-conjugated dUTP and reactionbuffer) for 90 min at 37° C. After removing the DNA labeling solution byrinsing cells with Rinsing Buffer, the cells were incubated with thePropidium Iodide/RNase A solution, incubated for 30 min at roomtemperature in the dark, and then analyzed by flow cytometry within 3 hof staining.

Caspase-3 Activity Assay.

To measure cell-free caspase-3 activity, whole cell extracts (20–30 μg)from untreated or treated LNCaP cells were incubated with 20 μM of thefluorogenic substrate caspase-3/caspase-7 (Ac-DEVD-AMC) for 30 min at37° C. in 100 μl of assay buffer (50 mM Tris, pH 8.0). Measurement ofthe hydrolyzed AMC groups was performed on a VersaFluor™ Fluorometer(Bio-Rad) as described previously (Nam S, Smith D M, Dou Q P., 2001,Ester bond-containing tea polyphenols potently inhibit proteasomeactivity in vitro and in vivo. J Biol Chem 276:13322–30).

Soft Agar Assay.

The soft agar assay was performed as described previously (Menter D G,Sabichi A L, Lippman S M., 2000, Selenium effects on prostate cellgrowth. Cancer Epidemiol Biomarkers Prev 9:1171–82) with a fewmodifications. In brief, in a six-well plate, a bottom feeder layer(0.6% agar) was prepared with DMEM media containing 10% fetal bovineserum, 100 units/ml penicillin, and 100 μg/ml streptomycin. A top layer(0.3% agar) was prepared with DMEM and the same media as described abovebut containing 2×10⁴ prostate cancer LNCaP cells and 50 μM of lactam 1or lactam 12, or equal volume of solvent (DMSO) as a control. Plateswere incubated at 37° C. in 5% CO₂ in a humidified incubator for threeweeks. MTT (1 mg/ml) was added to each well and incubated overnight toallow complete formation of purple formazan crystals. The plates werethen scanned and photographed, and the number of colonies was quantifiedby Quantity one v.4.0.3 software (Bio-Rad, Hercules, Calif.).

EXAMPLE 8 Screening for More Apoptotically Active Analogs of Lactam 1

Lactam 1 contains a chloro (Cl) group in the ortho position on thebenzene ring (FIG. 13A). To examine whether deletion or substitution ofthe Cl group could affect lactam 1 cell death-inducing ability,additional halogen (lactams 23, 46, and 49) and nonhalogen (lactam 47)analogs of lactam 1 were synthesized (FIG. 13A). These compounds werethen tested in the trypan blue dye exclusion assay, using lactam 1 as acomparison (FIG. 13B). Jurkat T cells were treated with each of thesecompounds at 50 μM for 24 h, followed by measurement of loss of cellmembrane permeability, indicative of a late apoptotic stage (FIG. 13B)(Wyllie A H, Kerr J F, Currie A R., 1980, Cell death: the significanceof apoptosis. Int Rev Cytol 68:251–306; Earnshaw W C., 1995, Nuclearchanges in apoptosis. Curr Opin Cell Biol 7:337–43). As a control,lactam 1 induced ˜52% cell death (FIG. 13B). Interestingly, removal ofthe Cl group from the benzene ring significantly decreased the celldeath-inducing activity to ˜25% (lactam 48; FIG. 13B). Furthermore,replacement of the Cl group with a smaller halogen atom (—F; lactam 49)also decreased the death-inducing activity (to ˜35%), while analogs witha larger halogen group (—Br and —I; lactams 23 and 46, respectively;FIG. 13A) increased the cell death rates to 55 and 60% (FIG. 13B). Thesedata indicate that the size of the group in the ortho position on thebenzene ring is important for the compound's cell death-inducingactivity. The analog with the —NO₂ group in the ortho position of thebenzene ring, lactam 47 (FIG. 13A), exhibited the strongest effect witha total of ˜94% cell death (FIG. 13B). Therefore, the order of potencyof the tested compounds was: H<F<Cl<Br<I<NO₂.

EXAMPLE 9 Lactam 1 Induces Apoptosis Preferentially in Leukemic Jurkat Tover Non-Transformed, Immortalized NK Cells

To determine whether lactam 1 was able to induce apoptosispreferentially in tumor/transformed vs. normal/non-transformed cells,human leukemic Jurkat T cells and immortalized, non-transformed NK cells(YT line) (Drexler H G, Matsuo A Y, MacLeod R A., 2000, Continuoushematopoietic cell lines as model systems for leukemia-lymphomaresearch. Leuk Res 24:881–911) were treated with lactam 1 in bothconcentration- and time-dependent experiments. Treatment of Jurkat Tcells with 10 μM of lactam 1 for 24 h induced apoptosis-specific PARPcleavage fragment p85 (FIG. 14A), whose levels were further increasedwhen 25 μM of lactam 1 was used (FIG. 14A). After treatment with 50 μMof lactam 1, PARP degradation was further increased, as evidenced by asignificant decrease in expression of intact PARP protein (FIG. 14A). Incontrast, no PARP cleavage was detectable in the YT cells aftertreatment with lactam 1 at even 50 μM (FIG. 14A).

In the kinetic experiment, both Jurkat T cells and YT cells were treatedwith 30 μM of lactam 1 for 3, 6, or 24 h. Again, PARP cleavage wasdetected in Jurkat T cells first at 3 h, which was then increased at 6 h(FIG. 14B). By 24 h, the levels of PARP/p85 fragments in Jurkat T cellswere decreased, probably again due to further degradation (FIG. 14B).Importantly, no PARP cleavage was observed in YT cells in the samekinetics experiment (FIG. 14B). To confirm the tumor cell-selectivekilling activity of lactam 1, a trypan blue dye exclusion assay wasperformed in the same kinetic experiment. After 24 h, there was 42% celldeath in the Jurkat T cells compared to 9% in YT cells (FIG. 14C).Furthermore, by using phase-contrast microscopy, more cell death wasobserved in Jurkat T cells than YT cells (FIG. 14D). Thus, lactam 1induced apoptotic cell death selectively in tumor cells overnon-transformed cells.

EXAMPLE 10 Lactam 47 has Enhanced Apoptosis-inducing Activity Specificto Jurkat T, but not Normal YT Cells

To determine if lactam 47 is capable of inducing apoptosis at lowerconcentrations than lactam 1, a dose-response experiment was performedwith both compounds. Jurkat T cells were treated with lactam 47 at 2,10, 25, and 50 μM for 24 h, using 50 μM of lactam 1 as a comparison.Again, treatment with lactam 1 caused ˜50% cell death, measured bytrypan blue exclusion assay (FIG. 15A). Under the same experimentalconditions, lactam 47 induced cell death in aconcentration-dependent-manner: 25% cell death at 10 μM, 45% at 25 μM,and 80–90% at 50 μM (FIG. 15A). Therefore, lactam 47 is ˜2-fold morepotent than lactam 1. This conclusion was further supported by PARPcleavage assay using lysates prepared after 12 h treatment (FIG. 15B).Cleavage of PARP occurred in lactam 47-treated cells in a dose-dependentmanner with the highest level of PARP cleavage observed at 50 μM (FIG.15B). The levels of PARP cleavage induced by 50 μM of lactam 1 wereequivalent to ˜50% of that by 50 μM of lactam 47 (FIG. 15B).

In the same experiment, when immortalized, non-transformed NK cells weretreated with lactam 47 (using lactam 1 as a control), neither cell death(FIG. 15C) nor PARP cleavage (FIG. 15D) were observed. Therefore, likelactam 1, lactam 47 also induced apoptotic cell death preferentially intumor cells over non-transformed cells.

To further compare the potency of lactams 1 and 47, Jurkat T cells weretreated with 25 μM of lactam 47 versus 50 μM of lactam 1 for 3, 6, 12,and 24 h, followed by determination of trypan blue incorporation andPARP cleavage. After 3 h, lactam 47 at 25 μM caused 15% versus 11% celldeath with lactam 1 at 50 μM (FIG. 16A). Similarly, at 6 h, 24% oftrypan blue-positive cells were found after 25 μM lactam 47 treatment,while only 20% observed in 50 μM lactam 1-treated cells (FIG. 16A). Onlyat later time points (12 and 24 b), lactam 1 at 50 μM was slightly morepotent than lactam 47 at 25 μM (FIG. 16A). Similar levels of cleavedPARP were observed in Jurkat T cells treated with either 25 μM of lactam47 or 50 μM of lactam 1 at each time point (FIG. 16B). Therefore, lactam47 is able to induce similar amounts of apoptosis in Jurkat T cells at aconcentration half of that of lactam 1.

Levels of sub-G₁ populations, as a measurement of cells with DNAfragmentation (An B, Goldfarb R H, Siman R, Dou Q P., 1998, Noveldipeptidyl proteasome inhibitors overcome Bcl-2 protective function andselectively accumulate the cyclin-dependent kinase inhibitor p27 andinduce apoptosis in transformed, but not normal, human fibroblasts. CellDeath Differ 5:1062–75), were examined in Jurkat T cells treated withlactam 47 or lactam 1. Treatment with 50 μM of lactam 47 increased thesub-G₁ populations by 34 and 57%, respectively, at 12 and 24 h (FIG.17A). In comparison, 50 μM of lactam 1 treatment for 12 and 24 h inducedsub-G₁ populations by 10 and 16%, respectively (Smith D M, Kazi A, SmithL, Long T E, Heldreth B, Turos, E, Dou Q P., 2002, A novel beta-lactamantibiotic activates tumor cell apoptotic program by inducing DNAdamage. Mol Pharmacol 61:1348–58), confirming the greater potency oflactam 47.

EXAMPLE 11 Lactam 47 is able to Induce DNA-Damage in Jurkat T Cells

Jurkat T cells were treated with 50 μM of lactam 47, followed byperformance of TUNEL assay, which detects DNA strand breaks (Smith D M,Kazi A, Smith L, Long T E, Heldreth B, Turos, E, Dou Q P., 2002, A novelbeta-lactam antibiotic activates tumor cell apoptotic program byinducing DNA damage. Mol Pharmacol 61:1348–58). A significant population(˜70%) of the cells exhibited DNA strand breaks after 3 h of incubationwith lactam 47 (FIG. 17B). A total of 82–90% of the cells wereTUNEL-positive after 12–24 h treatment with lactam 47 (FIG. 17B). Inthis experiment, 66% of TUNEL-positive cells were observed aftertreatment with 50 μM of lactam 1 for 24 h (data not shown). Thus, theincreased DNA-damaging capability of lactam 47 is most likelyresponsible for its enhanced cell death-inducing activity (FIGS. 13–16).

EXAMPLE 12 -Lactams 1 and 47 Induces Apoptosis and Inhibit ColonyFormation in Human Prostate Cancer Cells

To determine if lactam 1 and lactam 47 could also activate death programin solid tumor cells, human prostate cancer LnCaP cells were treated for48 h with lactam 47 at 2–25 μM or lactam 1 at 50 μM, followed bymeasurement of cell-free caspase-3 activity. A dose-dependent increasein caspase-3 induction was observed in LNCaP cells treated with lactam47: by 2-, 2.5- and 4.2-fold, respectively, at 2, 10 and 25 μM (FIG.18A). Treatment with 50 μM of lactam 1 also increased levels ofcaspase-3 activity by 2.5-fold over the control (FIG. 18A). These dataare consistent with the conclusion that lactam 47 has greaterapoptosis-inducing potency than lactam 1.

The in vivo effects of lactam 1 and lactam 47 in a soft agar assay thatmeasures the transforming activity of human tumor cells wasinvestigated. LNCaP cells were plated in soft agar along with 50 μM oflactam 1, 50 μM of lactam 47, or solvent (DMSO), followed by a 21day-incubation to allow for colony formation. The solvent (DMSO)-treatedplates allowed for the development of ˜500 colonies (FIGS. 18A and 18B).Lactam 1 inhibited 91%, and lactam 47 completely blocked (˜100%), colonyformation of LNCaP cells (FIGS. 18A and 18B). Therefore, both lactamsare able to inhibit the transformation capability of prostate cancercells.

EXAMPLE 13 Lactam 1 Selectively Inhibits Proliferation and InducesApoptosis in Transformed but not Normal WI-38 Cells

A normal human fibroblast cell line (WI-38) and its SV40-transformedderivative (VA-13) were tested to determine if lactam I couldselectively induce cell death in tumor cells versus normal cells. An MTTassay was performed in a 96-well plate, which measures cellularmitochondrial function and thereby cytotoxicity. Treatment with 50 μMlactam 1 for 6 or 24 hours completely inhibited growth of thetransformed VA-13 cells, compared to the same cells untreated (0 hour)or treated with DMSO (FIG. 19A). In contrast, the normal WI-38 cellscontinued to grow even after 24 hours treatment with 50 μM lactam 1,although such a treatment slightly inhibited the cell growth, comparedto the DMSO-treated WI-38 cells (FIG. 19A). By 120 hours, no viableVA-13 cells are found, whereas the population of WI-38 cells is doubled(data not shown).

To investigate whether selective inhibition of cell proliferation bylactam 1 is related to selective induction of apoptosis in thetransformed vs. normal WI-38 cells, caspase activation and apoptosiswere measured. Treatment of VA-13 cells with lactam 1 for up to 48 hoursactivated cell-free caspase-9 activity (FIG. 19B). However, no caspase-9activation was detected in normal WI-38 cells under the same treatment(FIG. 19B). Similarly, processing and thereby activation of caspase-3was detected only in the transformed, but not normal, human cells (FIG.19C).

Consistent with selective activation of caspases, detachment was foundonly in VA-13, but not WI-38 cells, after lactam 1 treatment; thedetached VA-13 cells revealed apoptotic nuclear condensation andfragmentation (FIG. 19D). Even the remaining attached VA-13 cells showedapoptotic nuclear morphology (FIG. 19D), indicating that apoptosistriggered detachment. In contrast, the treated WI-38 cells remainedattached with normal nuclei (FIG. 19D). These results suggest thatlactam 1 is able to selectively activate caspases and induce apoptosisin the transformed cells versus normal cells.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. An N-thiolated β-lactam compound having the structure of Formula A,

in which R₁ is a hydrocarbon group having 1–8 carbon atoms; R₃ is an —S-alkyl or —S-aryl wherein the alkyl or aryl group has 1–12 carbon atoms; and R₄ is cycloalkyl, heterocycloalkyl, cycloalkenyl, or heterocycloalkenyl, any of which can be optionally substituted with R₂, wherein R₂ is one or more halides, hydroxyl, nitro, cyano, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, alkoxy, amido, amino, —CO₂alkyl, wherein the alkyl group has 1–10 carbons, CHO, COOH, or COX, wherein X is Cl, F, Br, or I; or a pharmaceutically acceptable salt thereof.
 2. The compound according to claim 1, wherein R₁ is an alkyl group having 1–6 carbon atoms.
 3. The compound according to claim 1, wherein R₂ is a halide or halides and is selected from Cl, Br, or F.
 4. An N-thiolated β-lactam compound wherein the N-thiolated β-lactam compound is selected from the group consisting of:

or a pharmaceutically acceptable salt of any of said compounds.
 5. An N-thiolated β-lactam compound wherein the N-thiolated β-lactam compound is selected from the group consisting of:

or a pharmaceutically acceptable salt of any of said compounds.
 6. An N-thiolated β-lactam compound wherein the N-thiolated β-lactam compound is selected from the group consisting of:

or a pharmaceutically acceptable salt of any of said compounds.
 7. The compound according to claim 1, wherein R₁ is a methyl group.
 8. The compound according to claim 1, wherein R₄ is heterocycloalkyl or heterocycloalkenyl any of which can be substituted with R₂.
 9. The compound according to claim 2, wherein R₁ is a methyl group.
 10. The compound according to claim 2, wherein R₄ is heterocycloalkyl or heterocycloalkenyl any of which can be substituted with R₂.
 11. The compound according to claim 9, wherein R₂ is a halide or halides and is selected from Cl, Br, or F.
 12. The compound according to claim 10, wherein R₂ is a halide or halides and is selected from Cl, Br, or F.
 13. The compound according to claim 1, wherein R₃ is —S(CH₂)_(n)CH₃, wherein n=0 to
 3. 14. The compound according to claim 13, wherein R₃ is —SCH₃.
 15. The compound according to claim 1, wherein R₃ is —S(CH₂)_(n)phenyl, wherein n=0 to
 1. 16. The compound according to claim 15, wherein R₃ is —SCH₂phenyl.
 17. The compound according to claim 4, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 18. The compound according to claim 4, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 19. The compound according to claim 4, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 20. The compound according to claim 4, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 21. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 22. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 23. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 24. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 25. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 26. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 27. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 28. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 29. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 30. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 31. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 32. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 33. The compound according to claim 5, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 34. The compound according to claim 6, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof.
 35. The compound according to claim 6, wherein the N-thiolated β-lactam compound is:

or a pharmaceutically acceptable salt thereof. 